scholarly journals The de novo phospholipid effect of insulin is associated with increases in diacylglycerol, but not inositol phosphates or cytosolic Ca2+

1985 ◽  
Vol 231 (2) ◽  
pp. 269-278 ◽  
Author(s):  
R V Farese ◽  
J S Davis ◽  
D E Barnes ◽  
M L Standaert ◽  
J S Babischkin ◽  
...  
Keyword(s):  
Adipose Tissue ◽  
De Novo ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Specific Radioactivity ◽  
Phospholipid Content ◽  
Phospholipid Synthesis ◽  
Rat Adipose Tissue ◽  
Phosphatidylinositol 4 ◽  
Protein Kinase C Activation

We have previously reported that insulin increases the synthesis de novo of phosphatidic acid (PA), phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP), phosphatidylinositol 4,5-bisphosphate (PIP2) and diacylglycerol (DAG) in BC3H-1 myocytes and/or rat adipose tissue. Here we have further characterized these effects of insulin and examined whether there are concomitant changes in inositol phosphate generation and Ca2+ mobilization. We found that insulin provoked very rapid increases in PI content (20% within 15 s in myocytes) and, after a slight lag, PIP and PIP2 content in both BC3H-1 myocytes and rat fat pads (measured by increases in 32P or 3H content after prelabelling phospholipids to constant specific radioactivity by prior incubation with 32Pi or [3H]inositol). Insulin also increased 32Pi incorporation into these phospholipids when 32Pi was added either simultaneously with insulin or 1 h after insulin. Thus, the insulin-induced increase in phospholipid content appeared to be due to an increase in phospholipid synthesis, which was maintained for at least 2 h. Insulin increased DAG content in BC3H-1 myocytes and adipose tissue, but failed to increase the levels of inositol monophosphate (IP), inositol bisphosphate (IP2) or inositol trisphosphate (IP3). The failure to observe an increase in IP3 (a postulated ‘second messenger’ which mobilizes intracellular Ca2+) was paralleled by a failure to observe an insulin-induced increase in the cytosolic concentration of Ca2+ in BC3H-1 myocytes as measured by Quin 2 fluorescence. Like insulin, the phorbol diester 12-O-tetradecanoylphorbol 13-acetate (TPA) increased the transport of 2-deoxyglucose and aminoisobutyric acid in BC3H-1 myocytes. These effects of insulin and TPA appeared to be independent of extracellular Ca2+. We conclude that the phospholipid synthesis de novo effect of insulin is provoked very rapidly, and is attended by increases in DAG but not IP3 or Ca2+ mobilization. The insulin-induced increase in DAG does not appear to be a consequence of phospholipase C acting upon the expanded PI + PIP + PIP2 pool, but may be derived directly from PA. Our findings suggest the possibility that DAG (through protein kinase C activation) may function as an important intracellular ‘messenger’ for controlling metabolic processes during insulin action.

scholarly journals Phosphoinositide hydrolysis by guanosine 5′-[γ-thio]triphosphate-activated phospholipase C of turkey erythrocyte membranes

1988 ◽  
Vol 252 (2) ◽  
pp. 583-593 ◽  
Author(s):  
T K Harden ◽  
P T Hawkins ◽  
L Stephens ◽  
J L Boyer ◽  
C P Downes
Keyword(s):  
Phospholipase C ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Specific Radioactivity ◽  
Major Product ◽  
Erythrocyte Membranes ◽  
Low Concentrations ◽  
Phosphate Response ◽  
Phosphatidylinositol 4 ◽  
Rate Of Accumulation

Phosphatidylinositol (PtdIns), phosphatidylinositol 4-phosphate (PtdIns4P) and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] of turkey erythrocytes were labelled by using either [32P]Pi or [3H]inositol. Although there was little basal release of inositol phosphates from membranes purified from labelled cells, in the presence of guanosine 5′-[gamma-thio]triphosphate (GTP[S]) the rate of accumulation of inositol bis-, tris- and tetrakis-phosphate (InsP2, InsP3 and InsP4) was increased 20-50-fold. The enhanced rate of accumulation of 3H-labelled inositol phosphates was linear for up to 20 min; owing to decreases in 32P specific radioactivity of phosphoinositides during incubation of membranes with unlabelled ATP, the accumulation of 32P-labelled inositol phosphates was linear for only 5 min. In the absence of ATP and a nucleotide-regenerating system, no InsP4 was formed, and the overall inositol phosphate response to GTP[S] was decreased. Analyses of phosphoinositides during incubation with ATP indicated that interconversions of PtdIns to PtdIns4P and PtdIns4P to PtdIns(4,5)P2 occurred to maintain PtdIns(4,5)P2 concentrations; GTP[S]-induced inositol phosphate formation was accompanied by a corresponding decrease in 32P- and 3H-labelled PtdIns, PtdIns4P and PtdIns(4,5)P2. In the absence of ATP, only GTP[S]-induced decreases in PtdIns(4,5)P2 occurred. Since inositol monophosphate was not formed under any condition, PtdIns is not a substrate for the phospholipase C. The production of InsP2 was decreased markedly, but not blocked, under conditions where Ins(1,4,5)P3 5-phosphomonoesterase activity in the preparation was inhibited. Thus the predominant substrate of the GTP[S]-activated phospholipase C of turkey erythrocyte membranes is PtdIns(4,5)P2. Ins(1,4,5)P3 was the major product of this reaction; only a small amount of Ins(1:2-cyclic, 4,5)P3 was released. The effects of ATP on inositol phosphate formation apparently involve the contributions of two phenomena. First, the P2-receptor agonist 2-methylthioadenosine triphosphate (2MeSATP) greatly increased inositol phosphate formation and decreased [3H]PtdIns4P and [3H]PtdIns(4,5)P2 in the presence of a low (0.1 microM) concentration of GTP[S]. ATP over the concentration range 0-100 microM produced effects in the presence of 0.1 microM-GTP[S] essentially identical with those observed with 2MeSATP, suggesting that the effects of low concentrations of ATP are also explained by a stimulation of P2-receptors. Higher concentrations of ATP also increase inositol phosphate formation, apparently by supporting the synthesis of substrate phospholipids.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Breakdown of polyphosphoinositides and not phosphatidylinositol accounts for muscarinic agonist-stimulated inositol phospholipid metabolism in rat parotid glands

1983 ◽  
Vol 216 (3) ◽  
pp. 633-640 ◽  
Author(s):  
C P Downes ◽  
M M Wusteman
Keyword(s):  
Molecular Mechanisms ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Muscarinic Agonist ◽  
Parotid Glands ◽  
Muscarinic Agonists ◽  
Inositol 1 ◽  
Inositol 1,4,5 Trisphosphate ◽  
Rat Parotid ◽  
Phosphatidylinositol 4

The molecular mechanisms underlying the ability of muscarinic agonists to enhance the metabolism of inositol phospholipids were studied using rat parotid gland slices prelabelled with tracer quantities of [3H]inositol and then washed with 10 mM unlabelled inositol. Carbachol treatment caused rapid and marked increases in the levels of radioactive inositol 1-phosphate, inositol 1,4-bisphosphate, inositol 1,4,5-trisphosphate and an accumulation of label in the free inositol pool. There were much less marked changes in the levels of [3H]phosphatidylinositol, [3H]phosphatidylinositol 4-phosphate and [3H]phosphatidylinositol 4,5-bisphosphate. At 5 s after stimulation with carbachol there were large increases in [3H]inositol 1,4-bisphosphate and [3H]inositol 1,4,5-trisphosphate, but not in [3H]inositol 1-phosphate. After stimulation with carbachol for 10 min the levels of radioactive inositol 1,4-bisphosphate and inositol 1,4,5-trisphosphate greatly exceeded the starting level of radioactivity in phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate respectively. When carbachol treatment was followed by addition of sufficient atropine to block all the muscarinic receptors the radioactive inositol phosphates rapidly returned towards control levels. The carbachol-evoked changes in radioactive inositol phosphate and phospholipid levels were blocked in the presence of 2,4-dinitrophenol (an uncoupler of oxidative phosphorylation). The results suggest that muscarinic agonists stimulate a polyphosphoinositide-specific phospholipase C and that these lipids are continuously replenished from the labelled phosphatidylinositol pool. [3H]Inositol 1-phosphate in the stimulated glands probably arises via hydrolysis of inositol 1,4-bisphosphate and not directly from phosphatidylinositol.

scholarly journals Retinoic acid receptor γ 2 gene expression is up-regulated by retinoic acid in 3T3-L1 preadipocytes

1993 ◽  
Vol 293 (3) ◽  
pp. 807-812 ◽  
Author(s):  
Y Kamei ◽  
T Kawada ◽  
R Kazuki ◽  
E Sugimoto
Keyword(s):  
Adipose Tissue ◽  
Retinoic Acid ◽  
Mrna Expression ◽  
De Novo ◽  
Retinoic Acid Receptor ◽  
Mrna Level ◽  
Fold Increase ◽  
Dependent Manner ◽  
Alpha Beta ◽  
Rat Adipose Tissue

Retinoids, especially all-trans retinoic acid (RA), have been shown to inhibit the differentiation of preadipose cells. In the present study, the expression of retinoic acid receptors (RAR alpha, beta and gamma) and retinoid X receptors (RXR alpha, beta and gamma) was examined by Northern blot analysis in rat adipose tissue and mouse 3T3-L1 adipose cells. The adipose tissue and/or 3T3-L1 cells expressed mRNAs for a number of nuclear retinoid receptors, including RAR alpha, beta and gamma, and RXR alpha, beta and gamma. RAR alpha, RAR gamma, RXR alpha and RXR beta mRNAs were abundant in adipose tissue and 3T3-L1 cells. RXR gamma mRNA was detected in adipose tissue but not in 3T3-L1 cells. Treatment of 3T3-L1 cells with 1 microM RA led to a 4-5-fold increase in the RAR gamma mRNA level, but only a trace amount of RAR beta mRNA was detected. RAR gamma mRNA expression was rapidly (within 2 h) induced by physiological concentrations of RA in a dose-dependent manner. The response of RAR gamma mRNA expression to RA was reversible; rapid disappearance of RAR gamma mRNA occurred on RA removal. In addition, the induction of RAR gamma expression did not require de novo protein synthesis, but was completely abolished by an inhibitor of RNA synthesis. Using RAR gamma 1 and gamma 2 isoform-specific probes, the patterns of RAR gamma 1 and gamma 2 mRNA expression in 3T3-L1 cells in the presence and absence of RA were examined. RAR gamma 1 mRNA was detected in 3T3-L1 cells but was not affected by RA treatment; however, RAR gamma 2 mRNA was strongly induced by RA.

scholarly journals The labelling of polyphosphoinositides with [32P]Pi and the accumulation of inositol phosphates in vasopressin-stimulated hepatocytes

1986 ◽  
Vol 238 (2) ◽  
pp. 491-499 ◽  
Author(s):  
S Palmer ◽  
P T Hawkins ◽  
R H Michell ◽  
C J Kirk
Keyword(s):  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Water Soluble ◽  
Ionophore A23187 ◽  
Inositol Tetrakisphosphate ◽  
Inositol Phosphate Accumulation ◽  
Phosphatidylinositol 4 ◽  
Phosphate Groups

When hepatocytes were incubated with [32P]Pi, the kinetics for the labelling of the monoester phosphate groups of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate were similar to each other and slightly slower than that for the labelling of the gamma-phosphate of ATP. Analysis of the water-soluble 3H-labelled materials derived from [3H]inositol-labelled hepatocytes revealed that, in addition to inositol and its mono-, bis- and tris-phosphates (Ins, InsP, InsP2 and InsP3), these cells contained two unidentified radioactive compounds which co-eluted with InsP on anion-exchange chromatography. When [3H]inositol-labelled hepatocytes were stimulated with 0.23 microM-vasopressin in the presence of 10 mM-Li+, there was an accumulation of radioactivity in InsP, InsP2 and InsP3 but not in Ins or the two unidentified compounds. Further analysis of these inositol phosphates by h.p.l.c. revealed that vasopressin also stimulates the accumulation of inositol tetrakisphosphate (InsP4) in these cells. Vasopressin-stimulated InsP and InsP2 accumulations were maximal in the presence of 1-10 mM-Li+ but InsP3 accumulation continued to increase up to 50 mM-Li+. Accumulated inositol phosphates were retained within the cell. Li+ from 1 to 50 mM did not influence the extent of vasopressin-stimulated inositol lipid degradation in hepatocytes. In the absence of Li+, radioactivity in vasopressin-stimulated hepatocytes accumulated almost entirely in free inositol. The vasopressin-stimulated accumulation of inositol phosphates in the presence of 10 mM-Li+ was abolished by a V1-vasopressin antagonist. Inositol phosphate accumulation was not influenced by ionophore A23187, dimethyl sulphoxide or indomethacin.

scholarly journals Convulxin-induced platelet aggregation is accompanied by a powerful activation of the phospholipase C pathway

1994 ◽  
Vol 298 (1) ◽  
pp. 87-91 ◽  
Author(s):  
A Faili ◽  
J Randon ◽  
I M Francischetti ◽  
B B Vargaftig ◽  
M Hatmi
Keyword(s):  
Platelet Aggregation ◽  
Phospholipase C ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Platelet Activating Factor ◽  
Phosphatidyl Inositol ◽  
High Concentration ◽  
Adp Release ◽  
Pathway Formation ◽  
Phosphatidylinositol 4

Platelet aggregation and stimulation of phosphoinositide-specific phospholipase C (PLC) by thrombin and by convulxin (Cvx), a non-enzymic snake venom glycoprotein, were compared. Cvx-stimulated production of inositol phosphates by washed platelets was independent of the cyclo-oxygenase pathway, formation of platelet-activating factor and ADP release, but prostacyclin (prostaglandin I2), a stimulator of cyclic AMP formation, suppressed its effects on platelet and PLC activation. Kinetic analysis showed that inositol 1,4,5-trisphosphate formation reached its maximal value 15 s after platelet stimulation with Cvx and persisted for at least 5 min. Neomycin sulphate (10 mM), which complexes phosphatidylinositol 4-phosphate and phosphatidyl-inositol 4,5-bisphosphate, decreased the production of inositol phosphates, partially prevented platelet aggregation induced by a high concentration of Cvx (10 nM) and abolished both platelet aggregation and inositol phosphate formation induced by thrombin (2 units/ml) and by a stable prostaglandin H2 analogue, U46619 (1 microM). In contrast with neomycin sulphate, Na2SO4 had no significant effect against all agonists tested. It is concluded that platelet activation by Cvx is partially mediated by PLC and involves other mechanisms as well.

scholarly journals Receptor-mediated metabolism of the phosphoinositides and phosphatidic acid in rat lacrimal acinar cells

1984 ◽  
Vol 218 (1) ◽  
pp. 187-195 ◽  
Author(s):  
P P Godfrey ◽  
J W Putney
Keyword(s):  
Phosphatidic Acid ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Acinar Cells ◽  
Adrenergic Stimulation ◽  
Stimulus Response ◽  
Chemical Measurements ◽  
Apparent Loss ◽  
Lacrimal Acinar Cells ◽  
Phosphatidylinositol 4

The metabolism of the inositol lipids and phosphatidic acid in rat lacrimal acinar cells was investigated. The muscarinic cholinergic agonist methacholine caused a rapid loss of 15% of [32P]phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] and a rapid increase in [32P]phosphatidic acid (PtdA). Chemical measurements indicated that the changes in 32P labelling of these lipids closely resembled changes in their total cellular content. Chelation of extracellular Ca2+ with excess EGTA caused a significant decrease in the PtdA labelling and an apparent loss of PtdIns(4,5)P2 breakdown. The calcium ionophores A23187 and ionomycin provoked a substantial breakdown of [32P]PtdIns(4,5)P2 and phosphatidylinositol 4-phosphate (PtdIns4P); however, a decrease in [32P]PtdA was also observed. Increases in inositol phosphate, inositol bisphosphate and inositol trisphosphate were observed in methacholine-stimulated cells, and this increase was greatly amplified in the presence of 10 mM-LiCl; alpha-adrenergic stimulation also caused a substantial increase in inositol phosphates. A23187 provoked a much smaller increase in the formation of inositol phosphates than did either methacholine or adrenaline. Experiments with excess extracellular EGTA and with a protocol that eliminates intracellular Ca2+ release indicated that the labelling of inositol phosphates was partially dependent on the presence of extracellular Ca2+ and independent of intracellular Ca2+ mobilization. Thus, in the rat lacrimal gland, there appears to be a rapid phospholipase C-mediated breakdown of PtdIns(4,5)P2 and a synthesis of PtdA, in response to activation of receptors that bring about an increase in intracellular Ca2+. The results are consistent with a role for these lipids early in the stimulus-response pathway of the lacrimal acinar cell.

scholarly journals The effect of glucose, insulin and adrenaline on glycerol metabolism in vitro in rat adipose tissue

1976 ◽  
Vol 158 (2) ◽  
pp. 183-190 ◽  
Author(s):  
M C Domínguez ◽  
E Herrera
Keyword(s):  
Fatty Acids ◽  
Adipose Tissue ◽  
Specific Radioactivity ◽  
Glycerol Metabolism ◽  
Bicarbonate Buffer ◽  
Glycerol Utilization ◽  
Rat Adipose Tissue ◽  
Glycerol Uptake ◽  
Effect Of Glucose

The uptake and utilization of [1-14C]glycerol was determined in pieces of rat epididymal fat-pads incubated in Krebs--Ringer bicarbonate buffer containing albumin. Insulin (200 muunits/ml), adrenaline (epinephrine; 0.5 mug/ml) and glucose (0, 5, 15 and 20 mM) were added to the medium. Changes in the specific radioactivity of the tracer during the incubation were taken into account in calculating the rate of glycerol utilization. Adrenaline decreased glycerol uptake, whereas insulin plus adrenaline increased it. The rate of incorporation of glycerol into glycerides was decreased by adrenaline and insulin, singly or together. Insulin increased the rate of formation of CO2 and fatty acids from glycerol. The formation of CO2 and fatty acids was further enhanced by insulin plus adrenaline. The decrease in glycerol uptake induced by adrenaline, the decrease in incorporation of glycerol into glycerides induced by insulin and insulin plus adrenaline and the synthesis of fatty acids were dependent on the presence of glucose in the medium. Thus insulin and adrenaline act on glycerol utilization in adipose tissue and some of their effects are mediated by action on glucose metabolism, but others are independent of this.

scholarly journals Accumulation of the inositol phosphates in thrombin-stimulated, washed rabbit platelets in the presence of lithium

1984 ◽  
Vol 224 (2) ◽  
pp. 399-405 ◽  
Author(s):  
J D Vickers ◽  
R L Kinlough-Rathbone ◽  
J F Mustard
Keyword(s):  
Platelet Activation ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Important Change ◽  
Early Event ◽  
Rapid Degradation ◽  
Rabbit Platelets ◽  
Phosphatidylinositol 4 ◽  
Partial Release ◽  
Stimulation Of

Experiments with washed rabbit platelets demonstrate that stimulation with a low concentration of thrombin (0.1 unit/ml), that causes maximal aggregation and partial release of amine granule contents, also causes increased accumulation of [3H]inositol-labelled inositol trisphosphate (InsP3) in the presence of 20 mM-Li+. This concentration of Li+ was found to inhibit the degradation of inositol phosphates by phosphomonoesterases. This result indicates that phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] is degraded early after platelet stimulation with thrombin, although in a previous study we had found no decrease in amount. In the absence of Li+, the labelling of inositol bisphosphate (InsP2) increased more rapidly than that of InsP3, consistent with rapid degradation of InsP3 by phosphomonoesterase. After 30s the increase in InsP2 was augmented by Li+. This increase in InsP2 could have been due to increased degradation of phosphatidylinositol 4-phosphate or inhibition of breakdown of InsP2 to InsP with a lesser inhibition of breakdown of InsP3 to InsP2. The effect on InsP3 and InsP2 of stimulation of the platelets with 1.0 unit of thrombin/ml was comparable with the effect of the lower concentration of thrombin. Inositol phosphate (InsP) labelling did not increase in response to 0.1 unit of thrombin/ml, but increased when the platelets were stimulated with 1.0 unit of thrombin/ml. Whether the increase in InsP was due to increased degradation of phosphatidylinositol or a greater rate of breakdown of InsP2 to InsP than InsP to inositol cannot be determined in these experiments. These results indicate that degradation of PtdIns(4,5)P2 is an early event in platelet activation by thrombin and that formation of inositol phosphates and 1,2-diacylglycerol rather than a decrease in PtdIns(4,5)P2 may be the important change.

scholarly journals Interfacial hydrolysis of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate by turkey erythrocyte phospholipase C

1994 ◽  
Vol 298 (2) ◽  
pp. 499-506 ◽  
Author(s):  
S R James ◽  
R A Demel ◽  
C P Downes
Keyword(s):  
Phospholipase C ◽  
Surface Pressure ◽  
Inositol Phosphates ◽  
Specific Radioactivity ◽  
Initial Pressure ◽  
Maximum Activity ◽  
First Order Kinetics ◽  
High Specific Radioactivity ◽  
Phosphatidylinositol 4 ◽  
Hydrolysis Of

The activity of a beta-isoform of phospholipase C (PLC) partially purified from turkey erythrocyte cytosol was assayed using phospholipid monolayers formed at an air-water interface. PLC was rapidly purified at least 8000-fold by a sequence of ion-exchange, hydrophobic and heparin chromatographies. 33P-labelled substrates were prepared using partially purified PtdIns kinase and PtdIns4P 5-kinases, respectively, and purified by h.p.l.c. using an amino-cyano analytical column. Using such 33P-labelled phosphoinositides of high specific radioactivity, PLC activity was monitored directly by measuring the loss of radioactivity from monolayers as a result of the release of inositol phosphates and their subsequent dissolution and quenching in the subphase. Under these conditions, PtdIns4P hydrolysis obeyed approximately first-order kinetics whereas PtdIns(4,5)P2 hydrolysis was zero-order at least until 80% of the substrate had been degraded. PLC activity was markedly affected by the surface pressure of the monolayer, with reduced activity at extremes of initial pressure and with the most permissive pressures in the middle of the range investigated. The optimum surface pressure for hydrolysis of PtdIns4P was approx. 25 mN/m, but for PtdIns(4,5)P2 the maximum activity occurred at the markedly higher surface pressure of 30 mN/m. These data are discussed in terms of the substrate specificity and likely regulation of PLC beta isoforms engaged in degrading their substrate in biological membranes.

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