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Biomedical subjects

J M Mato

Publications and source records attributed to J M Mato.

At least 91 records · Page 5Linked to original sources
PubMed1990

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

We have investigated the topography of a glycosyl-phosphatidylinositol implicated in insulin action by a combination of two complementary methods: (a) chemical labelling with a non-permeable (isethionyl acetimidate) and a permeable (ethyl acetimidate) probe; and (b) enzymatic modifications with beta-galactosidase (EC 3.2.1.23) or phosphatidylinositol-specific phospholipase C (EC 3.1.4.3). Using the first approach the majority of the glycosyl-phosphatidylinositol is found in the outer surface of intact hepatocytes, adipocytes, fibroblasts and lymphocytes, but not in erythrocytes which presented only a 20% of the total labelled glycosyl-phosphatidylinositol to the exterior. Upon insulin addition (10 nM), about 60% of the total glycosyl-phosphatidylinositol was hydrolysed in both hepatocytes and adipocytes but not in erythrocytes. In agreement with the extracellular localization in hepatocytes and with the proposed role of this glycolipid in insulin action, treatment of rat hepatocytes with beta-galactosidase from Escherichia coli, an enzyme that hydrolyses the oligosaccharide moiety of the glycosyl-phosphatidylinositol, cleaved 65% of the total glycophospholipid and blocked the effect of insulin (but not of glucagon) on pyruvate kinase (EC 2.7.1.40). Similar treatment with phosphatidylinositol-specific phospholipase C from Bacillus cereus hydrolysed 62% of the total glycosyl-phosphatidylinositol. From the various approaches used it is concluded that the majority of this glycophospholipid is at the outer surface in a variety of insulin-sensitive cells.

Animals↗
PubMed1990

A phospho-oligosaccharide can reproduce the stimulatory effect of insulin on glycolytic flux in human fibroblasts.

It has been recently demonstrated that insulin promotes the hydrolysis of a glycosyl-phosphatidylinositol, stimulating the release of a phospho-oligosaccharide which displays several insulin-like effects. In the present study we have investigated whether the compound is able to mimic insulin action on glucose metabolism in human fibroblasts. Similarly to the hormone, the phospho-oligosaccharide elicited a dose dependent increase in lactate output and fructose 2,6-bisphosphate content. The effect of the compound was time dependent with a progressive increase starting from 2 hours of incubation. 1 microM phospho-oligosaccharide had half maximal effect on both parameters, increasing glycolytic flux by approximately 30% and fructose 2,6-bisphosphate content by 70%. Therefore the phospho-oligosaccharide appears to be able to strictly reproduce insulin action on glucose metabolism in human fibroblasts.

Cells, Cultured↗
PubMed1990

Effect of adrenalectomy and glucocorticoid treatment on the levels of an insulin-sensitive glycosyl-phosphatidylinositol in isolated rat hepatocytes.

Insulin resistance caused by dexamethasone administration to rats was accompanied by a marked decrease in the hepatocyte content of an insulin-sensitive glycosyl-phosphatidylinositol, as well as by a blockade of its hydrolysis in response to this hormone. In contrast, bilateral adrenalectomy provoked a significant increase of the cellular glycosyl-phosphatidylinositol levels. Under all the assayed metabolic conditions, a close direct correlation was established between the basal content of this compound and the number of insulin receptors present in the isolated hepatocytes.

Adrenal Glands↗
PubMed1990

Inactivation and dissociation of S-adenosylmethionine synthetase by modification of sulfhydryl groups and its possible occurrence in cirrhosis.

Catalytically active human and rat liver S-adenosylmethionine synthetase exists mainly in tetramer and dimer form. In liver biopsy samples from cirrhotic patients a marked reduction in total S-adenosylmethionine synthetase activity and a specific loss of the tetrameric form of the enzyme exist. We have investigated the possible role of sulfhydryl groups in maintaining the structure and activity of S-adenosylmethionine synthetase. Both forms of S-adenosylmethionine synthetase are rapidly inactivated by N-ethylmaleimide, and the loss of enzyme activity correlates with the incorporation of approximately 2 moles N-ethylmaleimide per mole of subunit. In addition, reaction with N-ethylmaleimide resulted in displacement of the tetramer-dimer equilibrium of the enzyme toward the dimer, but no monomer was detected under these conditions. A catalytically active monomeric S-adenosylmethionine synthetase was detected in the cytosolic extract from a liver biopsy sample from a cirrhotic patient, supporting our model for the structure of S-adenosylmethionine synthetase. Because treatment of S-adenosylmethionine synthetase with N-ethylmaleimide resembles the situation of this enzyme in cirrhotic patients, it is proposed that impaired protection of the enzyme from oxidizing agents caused by a decreased synthesis of glutathione can explain the diminished synthesis of S-adenosylmethionine in liver cirrhosis.

Animals↗
PubMed1990

Phospho-oligosaccharide dependent phosphorylation of ATP citrate lyase.

The effect of insulin on ATP citrate lyase phosphorylation has been shown to be mimicked by a phospho-oligosaccharide in intact adipocytes. We demonstrate that the addition of phospho-oligosaccharide to intact adipocytes enhances the phosphorylation of ATP citrate lyase in the same tryptic peptide as insulin does. The addition of phospho-oligosaccharide to an adipocyte extract also results in an increase in ATP citrate lyase phosphorylation but in a different site than that observed in intact cells. The phospho-oligosaccharide-dependent incorporation of phosphate into ATP citrate lyase in intact cells is resistant to isopropanol and acetic acid, but the phosphoenzyme phosphorylated in cell extracts is acid labile. In cell extracts, the addition of phospho-oligosaccharide markedly inhibits ATP hydrolysis, which may explain the effect of this molecule on ATP citrate lyase phosphorylation in broken cells. These results support the hypothesis that this phospho-oligosaccharide mediates some of the effects of insulin on protein phosphorylation. They also indicate that caution should be exercised in interpreting the results obtained by adding phospho-oligosaccharide to broken cell preparations.

ATP Citrate (pro-S)-Lyase↗
PubMed1990

Mechanisms and consequences of the impaired trans-sulphuration pathway in liver disease: Part I. Biochemical implications.

The energy-dependent conversion of methionine to S-adenosyl-L-methionine (SAMe) is catalysed by S-adenosyl-L-methionine synthetase (SAMe-synthetase) in the liver. In the hepatocyte, an equilibrium exists between the high and low molecular weight forms of SAMe-synthetase, which consist of a tetramer and a dimer, respectively, of a 48.5 kilodalton subunit. The 2 enzymic forms differ in their affinity for methionine and sensitivity to inhibition by pyrophosphate; 2 of the sulfhydryl groups of SAMe-synthetase have been identified as essential for the normal functioning of the enzyme. In patients with liver cirrhosis, a marked reduction in the utilisation of the high molecular weight SAMe-synthetase and displacement of the equilibrium occur, the molecular mechanism of which has yet to be established. This loss of activity is associated with a delay in methionine clearance and impairment of the trans-sulphuration pathway, which normally eliminates excess methionine by oxidising homocysteine to sulphate anion. It is hypothesised that in normal liver function the essential sulfhydryl groups of SAMe-synthetase are protected from oxidation by glutathione, a by-product of the trans-sulphuration pathway. However, glutathione levels are reduced in liver cirrhosis, and this may result in increased oxidation of the essential sulfhydryl groups, and consequent inactivation of the enzyme. Thus, the trans-sulphuration pathway may play an important role in the maintenance of normal SAMe-synthetase activity.

Humans↗
PubMed1990

Hydrolysis of glycosyl-phosphatidylinositol in response to insulin is reduced in cells bearing kinase-deficient insulin receptors.

A glycosyl-phosphatidylinositol (GPI) has been previously identified that serves as a precursor of the polar head group that mimics and may mediate some of the intracellular actions of insulin. Since many of the biological activities of insulin may depend upon the activity of the insulin receptor kinase, we evaluated the requirement for this activity in insulin-dependent GPI hydrolysis. For the analysis we used stably transfected CHO cell lines, expressing either the wild-type human insulin receptor or a mutant receptor that lacks tyrosine kinase activity (Chou et al., 1987) and a stably transfected CHO cell line, expressing the wild-type human insulin-like growth factor I (IGF-1) receptor (Steele-Perkins et al., 1988). A GPI was identified in both types of transfected cells and in both sets of parental cells by metabolic labeling with [3H]glucosamine or [3H]galactose. The isolated glycolipid was sensitive to hydrolysis by phospholipase C and to deamination by nitrous acid. Insulin induced a time- and dose-dependent hydrolysis of the GPI in the parental line and in the transfected cell types. Cells bearing normal human receptors hydrolyzed up to 70% of their radiolabeled GPI within 2 min of the addition of 0.1 nM insulin, whereas parental cells and cells expressing the mutant receptor hydrolyzed only 20-30% in response to 100 nM insulin. IGF-1 (5-50 nM) had little effect on GPI hydrolysis in these cells as well as in CHO cells expressing the human IGF-1 receptor. It is concluded that insulin-dependent GPI hydrolysis is mediated by the normal but not by a kinase-deficient insulin receptor.

Animals↗
PubMed1989

Insulin induces a similar reduction in the concentrations of its own receptor and of an insulin-sensitive glycosyl-phosphatidylinositol in isolated rat hepatocytes.

We have used isolated rat hepatocytes to study whether the insulin-induced reduction of its own receptors may modify the transduction of hormone signals by changes in the content of a glycosyl-phosphatidylinositol. Both subsequent insulin binding and glycosyl-phosphatidylinositol concentrations markedly decreased as a function of time and insulin concentration during preincubation of hepatocytes with insulin. The modifications observed in insulin binding were due to changes in receptor concentration. These results show that insulin regulates both the number of its own receptors and glycosyl-phosphatidylinositol concentrations in target cells, which may be of interest in many pathophysiological situations.

Animals↗
PubMed1989

Effect of a phospho-oligosaccharidic putative insulin messenger on insulin release in rats.

The phospho-oligosaccharide extracted from rat liver and supposed to act as the insulin second messenger inhibits glucose-stimulated insulin release. In the present study, this phospho-oligosaccharide was found not to affect D-[U-14C]glucose oxidation and 45Ca net uptake, but to inhibit insulin release evoked by either D-glucose or 2-ketoisocaproate in isolated rat islets. The relative extent of the latter inhibition was unaffected by either the concentration of D-glucose or the presence of dibutyryl-cyclic AMP, forskolin or glucagon in the incubation medium. At variance with the inhibitory effect of clonidine, that of the phospho-oligosaccharide was resistant to both blockade of alpha 2-adrenergic receptors or pre-treatment with the toxin of Bordetella pertussis. It is speculated, therefore, that such a phospho-oligosaccharide might interfere with a distal event in the insulin secretory sequence.

Animals↗
PubMed1989

Inhibition of proinsulin biosynthesis and conversion by a putative insulin mediator.

The phospho-oligosaccharide (POS), presumed to act at the postreceptor level as the insulin second messenger, was recently reported to inhibit glucose-stimulated insulin release from rat pancreatic islets. In the present study, POS was also found to inhibit glucose-stimulated proinsulin biosynthesis and conversion in rat islets. By comparison with prior findings on the effects of both exogenous insulin and anti-insulin serum upon proinsulin synthesis, these results argue against the view that insulin would normally exert a negative feedback control upon the biosynthetic and secretory activities of islet B-cells.

Animals↗
PubMed1988

Different phosphorylated forms of an insulin-sensitive glycosylphosphatidylinositol from rat hepatocytes.

Labeling with [3H]galactose was employed to isolate a glycosylphosphatidylinositol from rat hepatocytes which might be involved in the action of insulin. The polar head group of this glycosylphosphatidylinositol was generated by phosphodiesterase hydrolysis with a phosphatidylinositol-specific phospholipase C from Bacillus cereus. By Dowex AG1 x 8 chromatography the polar head group could be separated into three radioactive peaks eluting at 100 mM (peak I), 200 mM (peak II) and 500 mM (peak III) ammonium formate, respectively. Peak III was the most active as an inhibitor of the cAMP-dependent protein kinase. Treatment of peak III with alkaline phosphatase markedly reduced its activity on cAMP-dependent protein kinase. When peaks I, II or III were treated with alkaline phosphatase and analyzed again by Dowex AG1 x 8 chromatography, the radioactivity eluted with the aqueous fraction. The above results indicate that the polar head group of the insulin-sensitive glycosylphosphatidylinositol from rat hepatocytes exists in three different phosphorylated forms and that the biological activity of this molecule depends on its phosphorylation state.

Alkaline Phosphatase↗
PubMed1988

Regulation and function of an insulin-sensitive glycosyl-phosphatidylinositol during T lymphocyte activation.

A combination of metabolic labeling and chemical or enzymatic modification was employed to isolate and biochemically characterize a set of glycosyl-phosphatidylinositol (gly-PI) molecules synthesized by T lymphocytes. Gly-PI displayed unique patterns of synthesis following mitogen activation relative to the phosphoinositides and major structural lipids. The increase with time in gly-PI was paralleled by the appearance of insulin receptors. Gly-PI molecules were sensitive to hydrolysis by a PI-specific phospholipase C and were rapidly (15 sec) degraded in response to insulin binding. The product of this hydrolysis is believed to be a novel inositol phosphate-glycan (IP-gly) that was shown to inhibit the activity of a cAMP-dependent protein kinase. These results demonstrate that T cells contain a structurally related set of gly-PI molecules, at least one of which is sensitive to insulin and may function as a second messenger of hormone action.

Animals↗
PubMed1988

Localisation of the insulin-sensitive phosphatidylinositol glycan at the outer surface of the cell membrane.

A phosphatidylinositol-glycan has been implicated in the mechanism of action of insulin. Some of the actions of insulin may be mediated by the generation of the polar head group of this phosphatidylinositol-glycan. Localisation of the insulin-sensitive phosphatidylinositol-glycan was investigated by reacting the glycophospholipid with the imidoester [1-14C]-isethionyl acetimidate. The present results indicate that most of the insulin-sensitive phosphatidylinositol-glycan is localized at the plasma membrane of rat liver, being 85% of the glycophospholipid present at the outer surface of the cell. These results suggest a paracrine action of insulin.

Animals↗
PubMed1988

Inhibition of cyclic AMP-dependent protein kinase by the polar head group of an insulin-sensitive glycophospholipid.

A glycophospholipid has been purified from rat liver membranes and shown to copurify with an insulin-sensitive glycophospholipid isolated from H35 hepatoma cells. The polar head group of this glycophospholipid is a phospho-oligosaccharide generated by treatment with phosphatidylinositol-specific phospholipase C from Staphylococcus aureus. It has been proposed that this phospho-oligosaccharide, which is also generated in response to insulin, may play a role in insulin action. Incubation of the catalytic subunit of cyclic AMP-dependent protein kinase with this phospho-oligosaccharide inhibited the activity of the kinase to phosphorylate histone IIA, a purified preparation of phospholipid methyltransferase and kemptide, a phosphate-accepting peptide. Inhibition of kinase activity was dose-dependent and 50% inhibition of histone phosphorylation was demonstrated with a concentration of phospho-oligosaccharide of around 2 microM. This effect was demonstrated in the presence of ATP at concentrations up to 1 mM, indicating that the phospho-oligosaccharide acts at physiological concentrations of ATP and that it does not compete with this nucleotide for the same binding site in the kinase. Inhibition by the phospho-oligosaccharide of kinase activity could be reversed by dilution or dialysis and was not reproduced by up to 50 microM myo-inositol, glucosamine, galactose, myo-inositol 1-phosphate, glucosamine 1-phosphate, galactose 1-phosphate or phosphorylcholine. The inhibitory activity was resistant to mild acid treatment but was labile to treatment with alkali, exposure to nitrous acid or incubation with sodium periodate. The phospho-oligosaccharide had no effect on the phosphorylation of lysine-rich histone by rat brain protein kinase C and on the binding of cyclic AMP to a cyclic AMP-dependent protein kinase. In conclusion, the data in this study suggested that a phospho-oligosaccharide generated from an insulin-sensitive glycophospholipid may play a role in insulin action by modulating cyclic AMP-dependent protein kinase activity.

Animals↗
PubMed1987

A phospho-oligosaccharide mimics the effect of insulin to inhibit isoproterenol-dependent phosphorylation of phospholipid methyltransferase in isolated adipocytes.

Addition of isoproterenol to isolated rat adipocytes prelabeled with [32P]phosphate caused an increase in the phosphorylation and activation of phospholipid methyltransferase. 32P-Labeled phospholipid methyltransferase was recovered by immunoprecipitation and gel electrophoresis. Analysis of 32P-labeled peptides revealed one site of phosphorylation regulated by isoproterenol, and analysis of phosphoamino acids demonstrated that the incorporation of [32P]phosphate was on phosphoserine. Incubation of adipocytes with isoproterenol in the presence of insulin or a phospho-oligosaccharide inhibited the phosphorylation and activation of this enzyme. The inhibitory effect of insulin on the phosphorylation of phospholipid methyltransferase was reversible, and it was mimicked by a phospho-oligosaccharide. The phospho-oligosaccharide was generated by hydrolysis of an isolated glycophospholipid with phosphatidylinositol-specific phospholipase C from Staphylococcus aureus. The insulin-like effect of this phospho-oligosaccharide on the phosphorylation of phospholipid methyltransferase was demonstrated in isolated adipocytes, and the effect was abolished by treatment of the phospho-oligosaccharide with 10% NH4OH, nitrous acid, or sodium periodate. These data suggest that in intact adipocytes the effect of insulin to inhibit the phosphorylation/activation of phospholipid methyltransferase is mediated by a phospho-oligosaccharide generated by a phosphatidylinositol-specific phospholipase C.

Adipose Tissue↗
PubMed1987

A phospho-oligosaccharide mimics insulin action on glycogen phosphorylase and pyruvate kinase activities in isolated rat hepatocytes.

A phospho-oligosaccharide which is the polar head group of a novel insulin-sensitive glycophospholipid has recently been involved in insulin action. We have investigated the insulin-like effects of this phospho-oligosaccharide on both glycogen phosphorylase a and pyruvate kinase activities of hepatocytes incubated in the presence of glucagon (0.1 nM). Similarly to insulin, the phospho-oligosaccharide antagonized glucagon-dependent activation of glycogen phosphorylase, as well as the inactivation of pyruvate kinase caused by this hormone. The antagonistic action of the phospho-oligosaccharide on glucagon effects was dose-dependent. Furthermore, it partially antagonized glucagon-stimulated cyclic AMP levels. These results support the hypothesis that this phospho-oligosaccharide mediates at least some insulin actions in hepatocytes.

Animals↗