The function of glycosyl phosphoinositides in hormone action

The molecular events involved in the cellular actions of insulin remain unexplained. Some of the acute actions of the hormone may be due to the intracellular generation of a chemical substance which modulates certain enzyme activities. Such an enzyme-modulating substance has been identified as an inositol phosphate-glycan, produced by the insulin-sensitive hydrolysis of a glycosyl-phosphatidylinositol (glycosyl-PtdIns) precursor. This precursor glycolipid is structurally similar to the glycosyl-phosphoinositide membrane protein anchor. The exposure of fat, liver or muscle cells to insulin results in the hydrolysis of glycosyl-PtdIns, giving rise to the inositol phosphate glycan and diacylglycerol. This hydrolysis reaction is catalysed by a glycosyl-PtdIns-specific phospholipase C. This enzyme has been characterized and purified from a plasma membrane fraction of liver. This reaction also results in the acute release of certain glycosyl-PtdIns-anchored proteins from the cell surface. Elucidation of the functional role of glycosyl-phosphoinositides in the generation of second messengers or the release of proteins may provide further insights into the pleiotropic nature of insulin action.     

Activation of multiple signal transduction pathways by endothelin in cultured human vascular smooth muscle cells

Cellular responses to the vasoconstrictor peptide, endothelin, have been investigated in quiescent cultured human vascular smooth muscle cells (hVSMC). Endothelin caused intracellular alkalinization and activation of the protein synthetic enzyme S6-kinase, but such responses were not associated with any mitogenic effects of endothelin on hVSMC. In myo-[3H]inositol-prelabelled hVSMC endothelin elicited a rapid increase in inositol bis- and tris-phosphates and concomitant hydrolysis of polyphosphoinositol lipids. In [3H]arachidonate-prelabelled hVSMC endothelin promoted production of diacylglycerol, the early kinetics of which parallelled polyphosphoinositol lipid hydrolysis. Such phospholipase C activation by endothelin was sustained in hVSMC with accumulation of inositol polyphosphates being markedly protracted and the decay of diacylglycerol slow. Endothelin promoted extracellular release of [3H]arachidonate-labelled material from hVSMC which derived via deacylation of both phosphatidylinositol and phosphatidylcholine. This process was inhibited by phospholipase A2 and lipoxygenase inhibitors, but insensitive to phospholipase C and cyclooxygenase inhibitors. Endothelin-induced activation of phospholipase C and phospholipase A2 signal transduction pathways (EC50 approximately 5-8 nM for both) in hVSMC apparently proceed in an independent parallel manner rather than a sequential one.     

Insulin provokes co-ordinated increases in the synthesis of phosphatidylinositol, phosphatidylinositol phosphates and the phosphatidylinositol-glycan in BC3H-1 myocytes.

BC3H-1 myocytes were cultured in the presence of [3H]inositol or [3H]glucosamine during their entire growth cycle to ensure that all lipids containing inositol and glucosamine were labelled to isotopic equilibrium or maximal specific radioactivity. After such labelling, a lipid (or group of lipids), which was labelled with both inositol and glucosamine, was observed to migrate between phosphatidylinositol 4-phosphate and phosphatidylinositol (PI) in two different t.l.c. systems. Insulin provoked rapid, sizeable, increases in the inositol-labelling of this lipid (presumably a PI-glycan), and these increases were similar to those observed in PI and PI phosphates. Our results indicate that insulin provokes co-ordinated increases in the net synthesis de novo of PI and its derivatives, PI phosphates and the PI-glycan, in BC3H-1 myocytes. This increase in synthesis of PI may serve as the mechanism for replenishing the PI-glycan during stimulation of its hydrolysis by insulin. Moreover, increases in the content of the PI-glycan may contribute to increases in the generation of head-group 'mediators' during insulin action.     

The role of glycosyl-phosphoinositides in hormone action

Despite significant advances in the past few years on the chemistry and biology of insulin and its receptor, the molecular events that couple the insulin-receptor interaction to the regulation of cellular metabolism remain uncertain. Progress in this area has been complicated by the pleiotropic nature of insulin's actions. These most likely involve a complex network of pathways resulting in the coordination of mechanistically distinct cellular effects. Since the well-recognized mechanisms of signal transduction (i.e., cyclic nucleotides, ion channels) appear not to be central to insulin action, investigators have searched for a novel second messenger system. A low-molecular-weight substance has been identified that mimics certain actions of insulin on metabolic enzymes. This substance has an inositol glycan structure, and is produced by the insulin-sensitive hydrolysis of a glycosyl-phosphatidylinositol in the plasma membrane. This hydrolysis reaction, which is catalyzed by a specific phospholipase C, also results in the production of a structurally distinct diacylglycerol that may selectively regulate one or more of the protein kinases C. The glycosyl-phosphatidylinositol precursor for the inositol glycan enzyme modulator is structurally analogous to the recently described glycosyl-phosphatidylinositol membrane protein anchor. Preliminary studies suggest that a subset of proteins anchored in this fashion might be released from cells by a similar insulin-sensitive, phospholipase-catalyzed reaction. Future efforts will focus on the precise role of the metabolism of glycosyl-phosphatidylinositols in insulin action.     

Inositol Phospholipid Hydrolysis by Rat Sciatic Nerve Phospholipase C

Rat sciatic nerve cytosol contains a phosphodiesterase of the phospholipase C type that catalyzes the hydrolysis of inositol phospholipids, with preferences of phosphatidylinositol 4'-phosphate (PIP) greater than phosphatidylinositol (PI) much greater than phosphatidylinositol 4',5'-bisphosphate (PIP2), at a pH optimum of 5.5-6.0 and at maximum rates of 55, 13, and 0.7 nmol/min/mg protein, respectively. Analysis of reaction products by TLC and formate exchange chromatography shows that inositol 1,2-cyclic phosphate (83%) and diacylglycerol are the major products of PI hydrolysis. [32P]-PIP hydrolysis yields inositol bisphosphate, inositol phosphate, and inorganic phosphate, indicating the presence of phosphodiesterase, phosphomonoesterase, and/or inositol phosphate phosphatase activities in nerve cytosol. Phosphodiesterase activity is Ca2+-dependent and completely inhibited by EGTA, but phosphomonoesterase activity is independent of divalent cations or chelating agents. Phosphatidylcholine (PC) and lysophosphatidylcholine (lysoPC) inhibit PI hydrolysis. They stimulate PIP and PIP2 hydrolysis up to equimolar concentrations, but are inhibitory at higher concentrations. Both diacylglycerols and free fatty acids stimulate PI hydrolysis and counteract its inhibition by PC and lysoPC. PIP2 is a poor substrate for the cytosolic phospholipase C and strongly inhibits hydrolysis of PI. However, it enhances PIP hydrolysis up to an equimolar concentration.     

Platelet-activating factor-induced hydrolysis of phosphatidylinositol 4,5-bisphosphate stimulates the production of reactive oxygen intermediates in macrophages.

To investigate the relationship between inositol lipid hydrolysis and reactive oxygen-intermediate (ROI) production in macrophages we have examined the effect of platelet-activating factor (PAF) on normal bone marrow-derived macrophages. Addition of PAF to macrophages prelabelled with [3H]inositol caused a marked and rapid increase in [3H]inositol trisphosphate levels. Similarly when PAF was added to [3H]-glycerol prelabelled macrophages there was a rapid increase in 1,2-diacyl[3H]glycerol levels. These events preceded any increase in the rate of PAF-stimulated ROI production by a discernible period of several seconds. Increasing concentrations of PAF led to a markedly similar increase in both ROI production and [3H]inositol lipid hydrolysis suggesting that inositol lipid hydrolysis may lead to the generation of ROI in macrophages. Further evidence that this is the case came from experiments in which pretreatment of macrophages with phorbol esters was shown to inhibit both PAF-stimulated [3H]inositol phosphate production and ROI production to a markedly similar degree. Similarly pertussis toxin inhibited both PAF-stimulated ROI production and [3H]inositol phosphate production. Phorbol esters were shown to activate ROI production in normal bone marrow-derived macrophages whereas the Ca2+ ionophore, A23187, did not. These experiments suggest that PAF stimulates a pertussis toxin-sensitive activation of inositol lipid hydrolysis leading to the formation of inositol trisphosphate and diacylglycerol. The diacylglycerol formed can then activate protein kinase C leading to the stimulation of ROI production in normal bone marrow-derived macrophages.     

Studies on the enzymatic pathways of calcium ionophore-induced phospholipid degradation and arachidonic acid mobilization in peritoneal macrophages

Exposure of mouse peritoneal macrophages to ionophore A23187 caused a rapid and extensive Ca2+-dependent phospholipid degradation and mobilization of arachidonic acid. Phosphatidylinositol, phosphatidylcholine and phosphatidylethanolamine all contributed to the arachidonic acid release, although the ethanolamine phospholipids incorporated [3H]arachidonic acid more slowly during the prelabeling period, particularly the plasmalogen form. Several enzymatic pathways could be positively identified as contributing to the ionophore-induced phospholipid degradation by the use of several different radiolabeled phospholipid precursors: (i) a phospholipase A-mediated deacylation, (ii) a phosphodiesterase (phospholipase C) reaction, rapidly generating diacylglycerol units from inositol phospholipids, and (iii) enzymatic processes generating diacylglycerol and CDP- and phosphocholine/ethanolamine from phosphatidylcholine/ethanolamine. The diacylglycerol formed was in part phosphorylated and in part hydrolyzed to monoacylglycerol, with retention of its arachidonic acid. These, and other, results indicate that the Ca2+-ionophore activates several apparently distinct phospholipid-degrading processes, in contrast to stimuli acting via cellular receptors.     

Further studies on the mechanism of action of substance P in rat brain,involving selective phosphatidylinositol hydrolysis

We have suggested that substance P, in cerebral cortex, causes a phosphatidylinositol (PI) breakdown by a dual mechanism suggesting the involvement of either phospholipase A₂ or phospholipase C. We have presently characterized further these effects. Substance P (65 pM) provoked an increase in lysoPI concomitant with a decrease in PI level. This finding confirms the involvement of phospholipase A₂ activation. To study the involvement of phospholipase C in the action of higher doses (0.65 M) of the peptide, we used pulse-chase experiments (where phospholipid depletion was monitored) and short-term³²P-labeled slices (where phospholipid synthesis was studied). Substance P evoked an acceleration of both hydrolysis and resynthesis of PI as early as 15 s. A prolonged exposure (30 min) resulted in stimulation of PI hydrolysis without subsequent resynthesis. The peptide did not cause any effect on inositol 1,4-bisphosphate and inositol 1,4,5-trisphosphate. These alterations in PI metabolism take place simultaneously with a generation of diacylglycerol which showed two maxima at both indicated times.     

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

Abstract: Glycosyl phosphatidylinositols have been implicated in insulin signaling through their action as precursors of second messenger molecules in peripheral tissues. In the present study, cultured rat astrocytes were used to investigate whether glycosyl phosphatidylinositol might be involved in the mechanism of insulin signal transduction in neural cells. A glycosyl phosphatidylinositol sensitive to hydrolysis by both phosphatidylinositol-specific phospholipase C and glycosyl phosphatidylinositol-specific phospholipase D and to nitrous acid deamination was purified. When astrocytes were exposed to 10 n M insulin, a rapid and significant reduction in the content of glycosyl phosphatidylinositol was observed within 1–2 min. In addition, an inverse concentration-dependent relationship between glycosyl phosphatidylinositol and diacylglycerol levels was found, suggesting a phospholipase C-mediated hydrolysis of glycosyl phosphatidylinositol in response to insulin. The effects of insulin were mediated through its own receptors and not through insulin-like growth factor (IGF)-I and/or IGF-II receptors, as demonstrated by affinity cross-linking studies. Also, the effects of 5 n M IGF-I or 5 n M IGF-II on glycosyl phosphatidylinositol and diacylglycerol levels were different from those caused by insulin and were not essentially modified by pretreatment of the cells with either platelet-derived growth factor (PDGF) or epidermal growth factor (EGF). When cells were sequentially incubated with PDGF and EGF, a reduction in both glycosyl phosphatidylinositol and diacylglycerol contents was observed; the diacyl-glycerol but not the glycosyl phosphatidyl content was reversed after incubation with IGF-I, and especially with IGF-II, for 10 min. Despite the remarkable homology among insulin, IGF-I, and IGF-II, our results indicate that in astrocytes these compounds probably use different signal transduction pathways.     

Proposal for a pathway to mediate the metabolic effects of insulin

This review seeks to assemble recent discoveries about insulin receptor/kinase, guanine nucleotide-binding proteins, phosphatidyl inositol metabolism, and protein phosphatases to provide a mechanistic pathway by which insulin would alter carbohydrate and fat metabolism. It proposes a hypothetical chain of events that leads from the insulin receptor to protein phosphatase-1. The sequence starts with insulin binding to its receptor, activating the intrinsic receptor/kinase activity. The insulin receptor phosphorylates a guanine nucleotide-binding protein, which activates a particular phospholipase C. This in turn stimulates the production of two lipid-derived messengers: inositol-phospho-glucosamine and diacylglycerol. These messengers trigger the effects of insulin. The diacylglycerol produced by insulin is thought to be analogous to the diacylglycerol produced by alpha-adrenergic stimulation, which activates protein kinase C. Activation of this kinase could account for increases in phosphorylation of certain proteins. The inositol-phospho-glucosamine is the cytosolic messenger for insulin. One of the enzymes activated by insulin is protein phosphatase type-1. It is known that the phosphatase decreases phosphorylation of certain target enzymes. In response to insulin, activation of protein phosphatase type-1 occurs with a stable conformational change that may involve rearrangement of disulfide bonds. Rearrangement is either directly in response to the cytosolic messenger or is catalyzed by an isomerase activated by the insulin messenger. Ultimately, protein phosphatase type-1 and/or the disulfide isomerase may together mediate the pleiotropic effects of insulin on carbohydrate and fat metabolism.     

Mammalian phosphoinositide-specific phospholipase C isoenzymes

Procaryotic and eucaryotic cells have evolved multiple pathways for communication with their external environment. The inositol 1,4,5-trisphosphate/diacylglycerol second messenger system is an example of such a signal transduction pathway which is present in multicellular eucaryotic organisms. Binding of an agonist to a specific cell surface receptor promotes rapid hydrolysis of phosphatidylinositol 4,5-bisphosphate. The pivotal enzyme for this second messenger system is phosphoinositide-specific phospholipase C which hydrolyzes phosphatidylinositol 4,5-bisphosphate to generate the two second messengers, inositol 1,4,5-trisphosphate and diacylglycerol. Recently, much progress has been made in the purification, characterization and cDNA cloning of multiple PI-PLC isoenzymes. The results of the recent studies on phosphoinositide-specific phospholipase C are reviewed.     

PAF-induced activation of polyphosphoinositide-hydrolyzing phospholipase C in cerebral cortex.

The action of platelet-activating factor (PAF) on phosphoinositide hydrolysis was studied in rat brain slices. PAF produced a significant increase of 32P incorporation into phosphoinositides and phosphatidic acid (PA), in a dose- and time-dependent manner. Concomitantly, an increase of inositol phosphates and diacylglycerol (DAG) production was observed. Both inositol bisphosphate (IP2) and inositol trisphosphate (IP3) were detected as early as 5 s and they returned immediately to basal levels; concomitantly, formation of inositol monophosphate (IP) was detected. These findings demonstrated that PAF causes a rapid hydrolysis of polyphosphoinositides in cerebral cortex by a phospholipase C-dependent mechanism followed by subsequent resynthesis.     

Phosphatidylinositol-specific phospholipase C from Bacillus cereus combines intrinsic phosphotransferase and cyclic phosphodiesterase activities: a 31P NMR study

The inositol phosphate products formed during the cleavage of phosphatidylinositol by phosphatidylinositol-specific phospholipase C from Bacillus cereus were analyzed by 31P NMR. 31P NMR spectroscopy can distinguish between the inositol phosphate species and phosphatidylinositol. Chemical shift values (with reference to phosphoric acid) observed are 0.41, 3.62, 4.45, and 16.30 ppm for phosphatidylinositol, myo-inositol 1-monophosphate, myo-inositol 2-monophosphate, and myo-inositol 1,2-cyclic monophosphate, respectively. It is shown that under a variety of experimental conditions this phospholipase C cleaves phosphatidylinositol via an intramolecular phosphotransfer reaction producing diacylglycerol and D-myo-inositol 1,2-cyclic monophosphate. We also report the new and unexpected observation that the phosphatidylinositol-specific phospholipase C from B. cereus is able to hydrolyze the inositol cyclic phosphate to form D-myo-inositol 1-monophosphate. The enzyme, therefore, possesses phosphotransferase and cyclic phosphodiesterase activities. The second reaction requires thousandfold higher enzyme concentrations to be observed by 31P NMR. This reaction was shown to be regiospecific in that only the 1-phosphate was produced and stereospecific in that only D-myo-inositol 1,2-cyclic monophosphate was hydrolyzed. Inhibition with a monoclonal antibody specific for the B. cereus phospholipase C showed that the cyclic phosphodiesterase activity is intrinsic to the bacterial enzyme. We propose a two-step mechanism for the phosphatidyl-inositol-specific phospholipase C from B. cereus involving sequential phosphotransferase and cyclic phosphodiesterase activities. This mechanism bears a resemblance to the well-known two-step mechanism of pancreatic ribonuclease, RNase A.     

Oxytocin and vasopressin stimulate inositol phosphate production in human gestational myometrium and decidua cells

The involvement of phosphoinositide hydrolysis in the action of oxytocin and vasopressin on the uterus was investigated in gestational myometrium and decidua cells by measuring the production of inositol phosphates. Both peptides stimulated a dose related increase in all three inositol phosphates in myometrium. This may be related to the control of sarcoplasmic Ca⁺⁺ levels in the myometrium. Oxytocin and vasopressin also stimulated inositol 1-phosphate (IP) production in decidua cells. The hydrolysis of phosphatidylinositol by decidua homogenates exhibited a precursor-product relationship for diacylglycerol and arachidonic acid accumulation. Hence both peptides may mobilise free arachidonic acid, for prostaglandin biosynthesis, from decidua cell phosphoinositides by the sequential action of phospholipase C and diacylglycerol lipase.     

The catabolism of phosphatidylinisitol by an EDTA-insensitive phospholipase A1 and calcium-dependent phosphatidylinositol phosphodiesterase in rat brain

1. A rat brain supernatant and microsomal fraction contained a phospholipase A1 enzyme which hydrolysed phosphatidylinositol at pH 8 in the absence of calcium. Triolein and phosphatidylcholine were not attacked under the same incubation conditions. 2. No evidence could be obtained for a phospholipase A2 in the microsomal preparation, and in the presence of Ca2+ the release of fatty acid observed was due to phosphatidylinositol phosphodiesterase followed by diacylglycerol lipase action. 3. Brain phosphatidylinositol phosphodiesterase showed extensive activity in the alkaline range (7-8.5) as well as at pH 5-5.5. The activity at higher pH values required higher calcium concentrations and disappeared on purification of the soluble enzyme by ammonium sulphate fractionation. 4. In general the ratio between inositol 1,2-(cyclic)phosphate and inositol 1-phosphate produced by phosphodiesterase action decreased with increasing pH.     

Identification of a novel insulin-sensitive glycophospholipid from H35 hepatoma cells

This study identifies and partially characterizes an insulin-sensitive glycophospholipid in H35 hepatoma cells. The incorporation of [3H]glucosamine into cell lipids was investigated. A major labeled lipid was purified by sequential thin layer chromatography using first an acid followed by a basic solvent system. After hydrochloric acid hydrolysis and sugar analysis by thin layer chromatography, 80% of the radioactivity in the purified lipid was found to comigrate with glucosamine. H35 cells were prelabeled with [3H]glucosamine for either 4 or 24 h and treated with insulin causing a dose-dependent stimulation of turnover of the glycophospholipid which was detected within 1 min. The purified glycolipid was cleaved by nitrous acid deamination indicating that the glucosamine C-1 was linked to the lipid moiety through a glycosidic bond. [14C]Ethanolamine, [3H]inositol, and [3H]sorbitol were not incorporated into the purified glycolipid. The incorporation of various fatty acids into this glycolipid was also studied. [3H]Palmitate was found to be preferentially incorporated while myristic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, and arachidonic acid were either not incorporated or incorporated less than 10% of palmitate. The purified glycolipid labeled with [3H]palmitate was cleaved by treatment with phospholipase A2 but was resistant to mild alkali hydrolysis suggesting the presence of a 1-hexadecyl,2-palmitoyl-glyceryl moiety in the purified lipid. Treatment of labeled glycophospholipid with phosphatidylinositol-specific phospholipase C from Staphylococcus aureus generated a compound migrating as 1-alkyl,2-acyl-glycerol and a polar head group with a size in the range from 800 to 3500. These findings coupled with the nitrous acid deamination demonstrate that glucosamine was covalently linked through a phosphodiester bond to the glyceryl moiety of the purified glycolipid. These findings suggest that insulin acts on this glycophospholipid by stimulating an insulin-sensitive phospholipase C. This unique glycophospholipid may play an important role in insulin action by serving as precursor of insulin-generated mediators.     

Involvement of PL-D in the alternate signal tranduction pathway of macrophages induced by an exernal stimulus

The alternate pathway of signal transduction via hydrolysis of phosphatidylcholine, the major cellular phospholipid, has been investigated in murine peritoneal macrophages. A sustained formation of diacylglycerol, is preceded by an enhanced production of phosphatidic acid, when the macrophages were given a stimulus with 12-O-tetradecanoyl phorbol-13-acetate for sixty minutes. Production of choline and choline metabolites are significantly increased too. Propranolol, which inhibits phosphatidate phosphohydrolase, the enzyme responsible for conversion of phosphatidic acid to diacylglycerol, can effectively block the formation of diacylglycerol. Inhibition of protein kinase C either by its inhibitors, staurosporine and H-7 or by depletion, apparently affect the generation of the lipid products. Moreover, based on the results of transphosphatidylation reaction, involvement of a phospholipase D in the phosphatidylcholine-hydrolytic pathway in macrophages is predicted. These observations support the view that probably the phorbol ester acting directly on protein kinase C of the macrophages activate their phosphatidylcholine-specific phospholipase D to allow a steady generation of second messengers, to enable them to participate in the cell signalling process in a more efficient manner than those generated in the phosphoinositide pathway of signal transduction. (Mol Cell Biochem 000: 000-000,1999)     

A pertussis/cholera toxin sensitive G-protein may mediate vasopressin-induced inositol phosphate formation in smooth muscle cell

Arg-vasopressin (AVP) stimulates the production of inositol-1,4,5-triphosphate, inositol-1,4-bisphosphate and inositol-1-phosphate in A10 smooth muscle cell line. The AVP stimulation is rapid, time and dose dependent with an ED50 value of 5 nM. Protein kinase C activator, phorbol ester blocks the AVP effect on the production of inositol phosphates, suggesting that AVP induced phospholipase C (PLC) activation is under the negative feedback regulation by diacylglycerol production. Prolonged overnight treatment with either pertussis toxin and cholera toxin resulted partial inhibition of AVP-induced production of inositol phosphates. This result suggests that a novel G-protein similar to transducin might be involved in the AVP-induced PLC activation.     

Insulin-induced glycerolipid mediators and the stimulation of glucose transport in BC3H-1 myocytes

We have previously demonstrated that insulin stimulates glycerolipid synthesis and phospholipid hydrolysis in BC3H-1 myocytes, resulting in the generation of membrane diacylglycerol, a known cellular mediator. This led us to the original proposal that diacylglycerol may contribute to the mediation of insulin action, especially stimulation of glucose transport. The fact that agents such as phenylephrine and phorbol esters, which increase or act as membrane diacylglycerols, are fully active in stimulating glucose transport in this tissue lent further support to this proposal. In this paper, we demonstrate that the diacylglycerol analogues PMA (4 beta-phorbol 12-myristate 13-acetate) and mezerein (both possessing 12 beta- and 13 alpha-O-linked substituents as well as a 4 beta-hydroxyl group) each increase the Vmax of the glucose transporter as does insulin. Diacylglycerol generated by the addition of phospholipase C also stimulates glucose uptake to a maximum which is equal and nonadditive to that of insulin, while addition of the narrowly active phosphatidylinositol-specific phospholipase C which generates the putative phosphoinositol-glycan mediator of Saltiel et al. (Saltiel, A., Fox, J., She Lin, P., and Cutrecasas, P. (1986) Science 233, 967-972) stimulates pyruvate dehydrogenase in these cells without any effect on glucose uptake. Pretreatment of the myocytes with PMA resulted in desensitization of subsequent glucose uptake to stimulation by phenylephrine, but had no effect on stimulation of glucose uptake by phospholipase C or by insulin, indicating that PMA pretreatment primarily desensitizes agonist-induced polyphosphoinositide hydrolysis which, as we have previously shown, is not involved in the insulin-induced generation of diacylglycerol. This was confirmed by the absence of intracellular Ca2+ mobilization during insulin administration, as measured by the sensitive fluorescent probe fura-2 in attached monolayer BC3H-1 myocytes. Furthermore, we have shown that insulin-generated diacylglycerol satisfies several criteria for a mediator of insulin action, including the demonstration that insulin-stimulated endogenous diacylglycerol generation is antecedent to glucose transport and has an identical insulin dose-response curve and moreover that the magnitude and time course of subsequent stimulation of glucose transport is reproduced by the addition of the simple exogenous diacylglyerol, dioctanoylglycerol, in the complete absence of the hormone. These results establish a central role for insulin-induced glycerolipid metabolism in mediating insulin-stimulated glucose transport in BC3H-1 myocytes.     

Insulin effect on isolated rat hepatocytes: diacylglycerol-phosphatidic acid interrelationship.

It is widely accepted that insulin action does not involve inositol phospholipid hydrolysis through the stimulation of a phosphatidylinositol-specific phospholipase C (PI-PLC). This consideration prompted us to investigate the insulin effect on the mechanism leading to the accumulation of diacylglycerol (DAG) and phosphatidic acid (PA) in rat hepatocytes. Basically, insulin induces: (i) a significant increase of both [3H]glycerol and fatty acid labelling of DAG; (ii) a significant increase of PA labelling preceding DAG labelling and paralleled by a decrease of phosphatidylcholine (PC) labelling. These observations, which suggest an insulin-dependent involvement of a phospholipase D, are strengthened by the increase of PC-derived phosphatidylethanol in presence of ethanol. Finally, the observation that the PA levels do not return to basal suggests that other mechanisms different from PC hydrolysis, such as the stimulation of direct synthesis of PA, may be activated.