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

1988 ◽  
Vol 256 (1) ◽  
pp. 185-188 ◽  
Author(s):  
R V Farese ◽  
D R Cooper ◽  
T S Konda ◽  
G Nair ◽  
M L Standaert ◽  
...  
Keyword(s):  
Insulin Action ◽  
De Novo ◽  
Specific Radioactivity ◽  
Growth Cycle ◽  
Head Group ◽  
Isotopic Equilibrium ◽  
Phosphatidylinositol Phosphates ◽  
Phosphatidylinositol 4 ◽  
Stimulation Of

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.

scholarly journals The relationship of calcium to receptor-controlled stimulation of phosphatidylinositol turnover. Effects of acetylcholine, adrenaline, calcium ions, cinchocaine and a bivalent cation ionophore on rat parotid-gland fragments

1975 ◽  
Vol 148 (3) ◽  
pp. 479-485 ◽  
Author(s):  
L M Jones ◽  
R H Michell
Keyword(s):  
Parotid Gland ◽  
De Novo ◽  
Head Group ◽  
Ionophore A23187 ◽  
Bivalent Cation ◽  
Phosphatidylinositol Turnover ◽  
Rat Parotid Gland ◽  
Muscarinic Cholinergic ◽  
Rat Parotid ◽  
Stimulation Of

The possibility that Ca2+ ions are involved in the control of the increased phosphatidylinositol turnover which is provoked by alpha-adrenergic or muscarinic cholinergic stimulation of rat parotid-gland fragments has been investigated. Both types of stimulation provoked phosphatidylinositol breakdown, which was detected either chemically or radiochemically, and provoked a compensatory synthesis of the lipid, detected as an increased rate of incorporation of 32Pi into phosphatidylinositol. Acetylcholine had little effect on the incorporation of labelled glycerol, whereas adrenaline stimulated it significantly, but to a much lower extent than 32P incorporation: this suggests that the response to acetylcholine was entirely accounted for by renewal of the phosphorylinositol head-group of the lipid, but that some synthesis de novo was involved in the response to adrenaline. The responses to both types of stimulation, whether measured as phosphatidylinositol breakdown or as phosphatidylinositol labelling, occurred equally well in incubation media containing 2.5 mm-Ca2+ or 0.2 mm-EGTA [ethanedioxybis(ethylamine)-tetra-acetic acid]. Incubation with a bivalent cation ionophore (A23187) led to a small and more variable increase in phosphatidylinositol labelling with 32Pi, which occurred whether or not Ca2+ was available in the extracellular medium: this was not accompanied by significant phosphatidylinositol breakdown. Cinchocaine, a local anaesthetic, produced parallel increases in the incorporation of Pi and glycerol into phosphatidylinositol. This is compatible with its known ability to inhibit phosphatidate phosphohydrolase (EC 3.1.3.4) and increase phosphatidylinositol synthesis de novo in other cells. These results indicate that the phosphatidylinositol turnover evoked by alpha-adrenergic or muscarinic cholinergic stimuli in rat parotid gland probably does not depend on an influx of Ca2+ into the cells in response to stimulation. This is in marked contrast with the K+ efflux from this tissue, which is controlled by the same receptors, but is strictly dependent on the presence of extracellular Ca2+. The Ca2+-independence of stimulated phosphatidylinositol metabolism may mean that it is controlled through a mode of receptor function different from that which controls other cell responses. Alternatively, it can be interpreted as indicating that stimulated phosphatidylinositol breakdown is intimately involved in the mechanisms of action of alpha-adrenergic and muscarinic cholinergic receptor systems.

scholarly journals The phosphoinositides exist in multiple metabolic pools in rabbit platelets

1986 ◽  
Vol 238 (2) ◽  
pp. 411-417 ◽  
Author(s):  
J D Vickers ◽  
J F Mustard
Keyword(s):  
Phosphatidic Acid ◽  
Specific Radioactivity ◽  
Rabbit Platelets ◽  
Phosphatidylinositol 4 ◽  
Stimulation Of

The labelling of the phosphoinositides and phosphatidic acid in washed rabbit platelets incubated with [32P]phosphate or [3H]glycerol was studied in the presence of isotope and after unincorporated isotope had been removed. With both isotopes the increase in the specific radioactivity of phosphatidylinositol 4,5-bisphosphate (PIP2) lagged behind that of phosphatidylinositol 4-phosphate (PIP) but the specific radioactivity remained higher after unincorporated isotope had been removed. This result was consistent with the presence of a second pool of PIP2, which interconverted slowly with the pool of PIP2 which was in direct equilibrium with PIP, proposed to explain the increase in specific radioactivity of PIP2 which accompanies the decrease in amount of PIP2 at 10 s in ADP-stimulated platelets. In platelets labelled with [3H]glycerol, the specific radioactivity of PIP2 became higher than that of PIP and the specific radioactivity of PIP became higher than that of phosphatidylinositol (PI). These results were interpreted to indicate that there were two pools of PIP; of these the pool with the higher specific radioactivity was the precursor of PIP2. Similarly, two pools of PI were proposed. The presence of pools of the phosphoinositides with different specific radioactivities necessitates the measurement of chemical amount of these compounds when studying the effect of stimulation of the platelets, since changes in labelling may not accurately reflect changes in the amount of the phosphoinositides.

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 Insulin-stimulated α-(methyl)aminoisobutyric acid uptake in skeletal muscle. Evidence for a short-term activation of uptake independent of Na+ electrochemical gradient and protein synthesis

1988 ◽  
Vol 253 (3) ◽  
pp. 625-629 ◽  
Author(s):  
A Gumà ◽  
X Testar ◽  
M Palacín ◽  
A Zorzano
Keyword(s):  
Protein Synthesis ◽  
Amino Acid ◽  
Amino Acid Transport ◽  
Insulin Action ◽  
De Novo ◽  
Acid Transport ◽  
Electrochemical Gradient ◽  
Aminoisobutyric Acid ◽  
System A ◽  
Stimulation Of

1. The present study was designed to explore the mechanisms by which insulin stimulates system A of amino acid transport in extensor digitorum longus (EDL) muscles, by using a system A analogue, alpha-(methyl)aminoisobutyric acid (MeAIB). 2. Insulin stimulation of MeAIB uptake was noted after only 30 min of incubation and was maximal at 60 min. Kinetics of the insulin effect on MeAIB uptake were characterized by an increased Vmax. without modification of Km for MeAIB. 3. Incubation of EDL muscles with cycloheximide for 90 min did not modify MeAIB uptake in either the presence or the absence of insulin, indicating the independence of insulin action from protein synthesis de novo. Incubations for 180 min with cycloheximide caused a decrease in basal MeAIB uptake; however, the percentage stimulation of amino acid transport by insulin was unaltered. Basal MeAIB uptake was increased by incubation for 180 min, but under these conditions no change in the percentage effect of insulin was found. 4. Ouabain, gramicidin D, or both, markedly decreased basal MeAIB uptake by EDL muscle, but the percentage effect of insulin was unaltered. 5. We conclude that insulin action on amino acid transport through system A in muscle is rapid, is characterized by an increased Vmax., and is independent of protein synthesis de novo and the Na+ electrochemical gradient. Our data are compatible with insulin acting directly on the system A transporter.

scholarly journals Role of an acidic compartment in synthesis of disaturated phosphatidylcholine by rat granular pneumocytes

1982 ◽  
Vol 208 (3) ◽  
pp. 651-658 ◽  
Author(s):  
Avinash Chander ◽  
Aron B. Fisher ◽  
Jerome F. Strauss
Keyword(s):  
Fatty Acids ◽  
Oleic Acid ◽  
Palmitic Acid ◽  
De Novo ◽  
Lung Surfactant ◽  
Specific Radioactivity ◽  
Intact Cells ◽  
Subcellular Compartment ◽  
Surfactant Phospholipids ◽  
Stimulation Of

A possible role for an acidic subcellular compartment in biosynthesis of lung surfactant phospholipids was evaluated with granular pneumocytes in primary culture. Incubation with chloroquine (100μm) was used to perturb this compartment. With control cells, incorporation of [9,10-3H]palmitic acid into total lipids and into total phosphatidylcholines increased linearly with time up to 4h. Total incorporation into phosphatidylcholine during a 1h incubation was 999+85pmol of [9,10-3H]palmitic acid, 458±18pmol of [1-14C]oleic acid and 252±15pmol of [U-14C]glucose per μg of phosphatidylcholine phosphorus. The cellular content of either disaturated phosphatidylcholine or total phosphatidylcholines did not change during a 2h incubation with chloroquine. In the presence of chloroquine, the specific radioactivity of [3H]palmitic acid in disaturated phosphatidylcholine increased by 40%, and that of disaturated-phosphatidylcholine fatty acids from [U-14C]glucose increased by 125%. Incorporation of [1-14C]oleic acid into phosphatidylcholine was decreased by chloroquine by 79% and 33% in the presence or absence of palmitic acid respectively. Chloroquine stimulated phospholipase activity in intact cells, and in sonicated cells at pH4.0, but not at pH8.5. The observations indicate that chloroquine stimulates synthesis of disaturated phosphatidylcholine in granular pneumocytes from fatty acids, both exogenous and synthesized de novo, which can be due to stimulation of acidic phospholipase. This stimulation of acidic phospholipase A activity by chloroquine appears to be coupled to the synthesis of disaturated phosphatidylcholine, thereby enhancing remodelling of phosphatidylcholine synthesized de novo. Our findings, therefore, implicate the involvement of an acidic subcellular compartment in the remodelling pathway of disaturated phosphatidylcholine synthesis by granular pneumocytes.

Antiparasitic Treatment of Patients with P. falciparum Malaria Reduces the Ability of Patient Serum to Induce Tissue Factor by Decreasing NF-κB Activation

1995 ◽  
Vol 73 (01) ◽  
pp. 039-048 ◽  
Author(s):  
A Bierhaus ◽  
Ch J Hemmer ◽  
N Mackman ◽  
R Kutob ◽  
R Ziegler ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Falciparum Malaria ◽  
De Novo ◽  
Neutralizing Antibody ◽  
Mobility Shift ◽  
Before And After ◽  
Tf Gene ◽  
Electrophoretic Mobility Shift Assays ◽  
Antiparasitic Treatment ◽  
Stimulation Of

SummarySerum from patients with P. falciparum malaria at day 1 (pretherapy) induces tissue factor (TF) in cultured endothelial cells. TF induction depends on de novo transcription as shown in Nuclear Run On assays. Electrophoretic mobility shift assays demonstrated binding of AP-1 and NF- κB/Rel proteins to their recognition sites in the TF promotor. After therapy (day 28), stimulation of TF antigen by patient serum is reduced by 70%. When serum obtained before and after therapy was compared, a decrease of NF-κB activation was evident. Activation of NF-κB-like proteins was in part dependent on TNFα in patient serum, since a TNFα neutralizing antibody reduced induction of TF transcription and translation and induction of NF-κB-like proteins. Induction of TF activity was suppressed by pDTC, an inhibitor of NF-κB activation. When different promotor constructs of the TF gene were tested, induction was dependent upon the presence of the intact NF-κB-like binding site in the TF promotor. A mutant with deleted NF-κB, but intact AP-1 sites was not inducible. Mutation of the AP-1 sites did not prevent induction, but reduced inducibility by pretherapy serum. Therefore, NF-κB/Rel proteins are responsible for induction of TF transcription by pretherapy serum, but AP-1 is needed for highest inducibility. The effect of antiparasitic therapy on the induction of TF by serum from patients with complicated P. falciparum malaria is dependent on a therapy-mediated loss of activation of NF-κB-like proteins in post-treatment patient serum.

scholarly journals Oestradiol inhibits collagen breakdown in the involuting rat uterus

1972 ◽  
Vol 127 (4) ◽  
pp. 705-713 ◽  
Author(s):  
Janet N. Ryan ◽  
J. Frederick Woessner
Keyword(s):  
Cathepsin D ◽  
Density Gradient Centrifugation ◽  
Post Partum ◽  
Gradient Centrifugation ◽  
Specific Radioactivity ◽  
Rat Uterus ◽  
Oestradiol Treatment ◽  
Cathepsin D Activity ◽  
Stimulation Of

1. The earlier observation (Woessner, 1969) of oestradiol inhibition of collagen breakdown is confirmed and extended. Administration of 100μg of oestradiol-17β/day to parturient rats strongly inhibits the loss of collagen from the involuting uterus. Three experiments show that this effect is due to an inhibition of collagen degradation rather than to a stimulation of collagen synthesis. 2. Uterine collagen was labelled with hydroxy[14C]-proline by the administration of [14C]proline near the end of pregnancy. By 3 days post partum, control uteri lost 83% of their collagen and 90% of their hydroxy[14C]proline. Uteri from oestradiol-treated rats lost only 50% of both total and labelled hydroxyproline, with no decrease in the specific radioactivity of the hydroxyproline. 3. Incorporation of [14C]proline into uterine collagen hydroxyproline in vivo was not affected by oestradiol treatment. 4. Urinary excretion of hydroxyproline was increased in post-partum control rats and decreased in oestradiol-treated rats. 5. An enzyme capable of cleaving 4-phenylazobenzyloxycarbonyl-l-prolyl-l-leucylglycyl- l-prolyl-d-arginine (a substrate for clostridial collagenase) increased in activity in the post-partum uterus and was unaffected by oestradiol treatment. 6. Uterine homogenates digested uterine collagen extensively at pH3.2. This digestion was unaffected by the oestradiol treatment. 7. Lysosomal fractions prepared by density-gradient centrifugation of uterine homogenates contained coincident peaks of cathepsin D activity and peptide-bound hydroxyproline. The cathepsin D and hydroxyproline contents of this peak were unaffected by oestradiol treatment.

scholarly journals β-Adrenergic stimulation alters oligosaccharide pyrophosphoryl dolichol metabolism in rat parotid acinar cells

1985 ◽  
Vol 231 (2) ◽  
pp. 431-438 ◽  
Author(s):  
S R Grant ◽  
E E Kousvelari ◽  
D K Banerjee ◽  
B J Baum
Keyword(s):  
Half Life ◽  
Specific Radioactivity ◽  
Acinar Cells ◽  
Protein Glycosylation ◽  
Significant Enhancement ◽  
Adrenergic Stimulation ◽  
Parotid Acinar Cells ◽  
Secretory Glycoproteins ◽  
Rat Parotid ◽  
Stimulation Of

beta-Adrenergic stimulation of rat parotid acinar cells markedly increases [3H]mannose incorporation into N-linked glycoproteins [Kousvelari, Grant, Banerjee, Newby & Baum (1984) Biochem. J. 222, 17-24]. More than 90% of this protein-bound [3H]mannose was preferentially incorporated into four secretory glycoproteins. The ratio of [3H]mannose/[14C]leucine present in these individual proteins was 1.7-4-fold greater with isoproterenol-treated cells than with untreated controls. In isoproterenol-stimulated cells, [3H]mannose incorporation into mannosylphosphoryl dolichol and oligosaccharide-PP-dolichol was increased 2-3-fold over that observed in unstimulated cells. Similarly, formation of mannosylated oligosaccharide-PP-dolichol was increased approx. 4-fold in microsomes prepared from isoproterenol-treated cells. Also, turnover of oligosaccharide-PP-dolichol was significantly increased (5-fold) by β-adrenergic stimulation; the half-life for oligosaccharide-PP-dolichol decreased from 6 min in control cells to 1.2 min in isoproterenol-stimulated cells. By 15 min after isoproterenol addition to acinar cells, the specific radioactivity of parotid oligosaccharide moieties increased about 3-fold over the value observed in the absence of the agonist. Taken together, these results strongly suggest that elevation of N-linked protein glycosylation in rat parotid acinar cells after β-adrenoreceptor stimulation resulted from significant enhancement in the synthesis of mannosylphosphoryl dolichol and oligosaccharide-PP-dolichol and the turnover of oligosaccharide-PP-dolichol.

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