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M F Vincent

Publications and source records attributed to M F Vincent.

At least 37 records · Page 2Linked to original sources
PubMed1997

Identification of new medium-chain acylcarnitines present in normal human urine.

Analysis of urinary medium-chain acylcarnitines extracted on C18 cartridges and gas chromatography mass spectrometry of their fatty acid moieties as picolinyl esters allowed the determination of the chemical structure of previously unidentified acylcarnitines in normal human urine. These are the 2,6-dimethylheptanoyl-, the 2,6-dimethyl-5-heptenoyl-, and the trans- and cis-3,4-methylene heptanoylcarnitines, also named 3-cyclopropane octanoylcarnitines. Assessment of the structure of these cyclopropane derivatives was obtained by 1H and 13C nuclear magnetic resonance spectroscopy. In addition, other acylcarnitines were tentatively identified.

Acylation↗
PubMed1997

Inborn errors of the purine nucleotide cycle: adenylosuccinase deficiency.

Adenylosuccinase catalyses two reactions in purine metabolism: the conversion of succinylaminoimidazole carboxamide ribotide (SAICAR) into aminoimidazole carboxamide ribotide (AICAR) along the de novo synthesis of purine nucleotides, and the conversion of adenylosuccinate (S-AMP) into AMP in the conversion of IMP into AMP. The hallmarks of adenylosuccinase deficiency are the presence of succinylaminoimidazole carboxamide riboside (SAICAriboside) and succinyladenosine (S-Ado) in body fluids. These normally undetectable succinylpurines are the products of the dephosphorylation, by cytosolic 5'-nucleotidase, of the two substrates of adenylosuccinase. The clinical picture of the enzyme deficiency is markedly heterogeneous with, as a rule, a profound, but nevertheless variable degree of psychomotor delay, often convulsions and/or autistic features, sometimes growth retardation and muscular dystrophy. The diagnostic tests that can be used for diagnosis, the enzyme and gene defects that have been identified, and the hypotheses that have been put forward to explain the pathophysiology of the disorder are reviewed.

Adenylosuccinate Lyase↗
PubMed1996

Substrate cycling between 5-amino-4-imidazolecarboxamide riboside and its monophosphate in isolated rat hepatocytes.

AICA (5-amino-4-imidazolecarboxamide)-riboside is taken up by isolated rat hepatocytes and converted by adenosine kinase (ATP:adenosine 5'-phosphotransferase, EC 2.7.1.20) into AICAR (ZMP), an intermediate of the de novo synthesis of purine nucleotides. We investigated if, in these cells, a cycle analogous to the adenosine-AMP substrate cycle operates between AICAriboside and ZMP. When 50 microM ITu, an inhibitor of adenosine kinase, was added to hepatocytes that had metabolized AICAriboside for 30 min, the concentration of ZMP decreased immediately. This was mirrored by a reincrease of AICAriboside. Rates of the ITu-induced decrease of ZMP and the increase of AICAriboside, calculated at different concentrations of ZMP, were first order, up to the highest concentration of ZMP (approx. 5 mumol/g of cells). Dephosphorylation of ZMP added to crude cytosolic extracts of rat liver displayed hyperbolic kinetics, with a Vmax of 0.65 mumol/min per g protein and an apparent Km of 5 mM, and was markedly inhibited by Pi, an inhibitor of IMP-GMP 5'-nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5). We conclude that hepatocyte ZMP is continuously dephosphorylated, most likely by IMP-GMP 5'-nucleotidase, into AICAriboside, which is rephosphorylated into ZMP by adenosine kinase. Substrate cycling was also shown to occur between other nucleoside analogs and their phosphorylated counterparts.

Aminoimidazole Carboxamide↗
PubMed1996

Stimulation of rat liver AMP-activated protein kinase by AMP analogues.

Stimulation of AMP-activated kinase (AMP-PK) by ZMP (5-amino-4-imidazolecarboxamide ribotide, AICAR), formed by adenosine kinase upon addition of AICAriboside to isolated rat hepatocytes, results in inhibition of fatty acid and cholesterol synthesis by inactivation of acetyl-CoA carboxylase and 3-hydroxy-3-methylglutaryl-CoA reductase, respectively (Henin et al. (1995) FASEB J. 9, 541-546). The effects of ZMP and other AMP analogues have now been compared with those of AMP on AMP-PK purified from rat liver. ZMP stimulated AMP-PK to the same maximal extent as AMP (about 10-fold). ZMP had less affinity for AMP-PK than AMP, but this affinity was similarly influenced by ATP: half-maximal effects, requiring 0.4 mM AMP or 5 mM ZMP at 3 mM ATP, were obtained with 9 microM AMP or 0.4 mM ZMP at 0.2 mM ATP. The kinetic parameters of AMP-PK for the SAMS peptide and for ATP were influenced in the same way by ZMP and AMP. Stimulation of AMP-PK by ZMP was additive with AMP, up to when maximal stimulation was obtained. Taken together, these results indicate that ZMP binds to the same site as AMP on AMP-PK. Tubercidin 5'-monophosphate, 2'-deoxy-AMP and Ara-AMP stimulated AMP-PK, but N6-methyl-AMP, 1,N6-etheno-AMP, 6-mercaptopurine riboside 5'-monophosphate, adenylosuccinate and succinyl-AICAR were ineffective, suggesting that a free 6-NH2 group may be important for binding of effectors to AMP-PK.

AMP-Activated Protein Kinases↗
PubMed1996

Hypoglycaemic effect of AICAriboside in mice.

We have previously demonstrated that in isolated hepatocytes from fasted rats, AICAriboside (5-amino 4-imidazolecarboxamide riboside), after its conversion into AICAribotide (AICAR or ZMP), exerts a dose-dependent inhibition on fructose-1,6-bisphosphatase and hence on gluconeogenesis. To assess the effect of AICAriboside in vivo, we measured plasma glucose and liver metabolites after intraperitoneal administration of AICAriboside in mice. In fasted animals, in which gluconeogenesis is activated, AICAriboside (250 mg/kg body weight) induced a 50% decrease of plasma glucose within 15 min, which lasted about 3 h. In fed mice, glucose decreased by 8% at 30 min, and normalized at 1 h. Under both conditions, ZMP accumulated to approximately 2 mumol/g of liver at 1 h. It decreased progressively thereafter, although much more slowly in the fasted state. Inhibition of fructose-1,6-bisphosphatase was evidenced by time-wise linear accumulations of fructose-1,6-bisphosphate, from 0.006 to 3.9 mumol/g of liver at 3 h in fasted mice, and from 0.010 to 0.114 mumol/g of liver at 1 h in fed animals. AICAriboside did not significantly influence plasma insulin or glucose utilization by muscle. We conclude that in vivo as in isolated hepatocytes, AICAriboside, owing to its conversion into ZMP, inhibits fructose-1,6-bisphosphatase and consequently gluconeogenesis.

Adenosine Triphosphate↗
PubMed1995

Cell-type specificity of inhibition of glycolysis by 5-amino-4-imidazolecarboxamide riboside. Lack of effect in rabbit cardiomyocytes and human erythrocytes, and inhibition in FTO-2B rat hepatoma cells.

The nucleoside AICAriboside (5-amino-4-imidazolecarboxamide riboside) has been shown to inhibit glycolysis in isolated rat hepatocytes [Vincent, Bontemps and Van den Berghe (1992) Biochem. J. 281, 267-272]. The effect is mediated by AICA-ribotide (ZMP), the product of the phosphorylation of AICA-riboside by adenosine kinase. To assess the cell-type specificity of the effect, studies were conducted in rabbit cardiomyocytes, human erythrocytes and rat hepatoma FTO-2B cells. AICA-riboside had no effect on glycolysis in cardiomyocytes, and a slight stimulatory effect in erythrocytes, but inhibited glycolysis by 65% at 250 microM concentration in FTO-2B cells, although only when tissue-culture medium was replaced by Krebs-Ringer bicarbonate buffer. At 500 microM AICAriboside, ZMP remained undetectable in cardiomyocytes, but reached 0.65 mM in erythrocytes and 5 mM in FTO-2B cells. In the latter, AICAriboside provoked up to 2-fold elevations of glucose 6-phosphate and fructose 6-phosphate, accompanied by a decrease in fructose 1,6-bisphosphate. This indicated inhibition of 6-phosphofructo-1-kinase (PFK-1). Accordingly, in FTO-2B cell-free extracts, the activity of PFK-1, measured under physiological conditions, was inhibited by approx. 70% by 5 mM ZMP. ZMP had a less pronounced effect on the activity of PFK-1 in normal rat liver; it did not influence the activity of PFK-1 in rat muscle, rabbit heart and human erythrocytes. It is concluded that the inhibitory effect of AICAriboside on glycolysis is dependent on both (1) the capacity of the cells to accumulate ZMP and (2) the presence of target enzymes which are sensitive to ZMP.

Adenosine Kinase↗
PubMed1995

Inhibition of fatty acid and cholesterol synthesis by stimulation of AMP-activated protein kinase.

AMP-activated protein kinase is a multisubstrate protein kinase that, in liver, inactivates both acetyl-CoA carboxylase, the rate-limiting enzyme of fatty acid synthesis, and 3-hydroxy-3-methyl-glutaryl-CoA reductase, the rate-limiting enzyme of cholesterol synthesis. AICAR (5-amino 4-imidazolecarboxamide ribotide, ZMP) was found to stimulate up to 10-fold rat liver AMP-activated protein kinase, with a half-maximal effect at approximately 5 mM. In accordance with previous observations, addition to suspensions of isolated rat hepatocytes of 50-500 microM AICAriboside, the nucleoside corresponding to ZMP, resulted in the accumulation of millimolar concentrations of the latter. This was accompanied by a dose-dependent inactivation of both acetyl-CoA carboxylase and 3-hydroxy-3-methylglutaryl-CoA reductase. Addition of 50-500 microM AICAriboside to hepatocyte suspensions incubated in the presence of various substrates, including glucose and lactate/pyruvate, caused a parallel inhibition of both fatty acid and cholesterol synthesis. With lactate/pyruvate (10/1 mM), half-maximal inhibition was obtained at approximately 100 microM, and near-complete inhibition at 500 microM AICAriboside. These findings open new perspectives for the simultaneous control of triglyceride and cholesterol synthesis by pharmacological stimulators of AMP-activated protein kinase.

AMP-Activated Protein Kinases↗
PubMed1993

Mechanisms of elevation of adenosine levels in anoxic hepatocytes.

Previous work has shown that normoxic isolated rat hepatocytes continuously produce adenosine from AMP and that the nucleoside is not catabolized further but immediately rephosphorylated by adenosine kinase [Bontemps, Van den Berghe and Hers (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 2829-2833]. We now report the effect of anoxia on adenosine production and on the AMP/adenosine substrate cycle. In cell suspensions incubated in O2/CO2, the adenosine concentration was about 0.4 microM. It increased 30-fold in cells incubated in N2/CO2 or with 5 mM KCN, and 20-fold in cells incubated with 2 mM amytal. Adenosine production, measured in hepatocytes in which adenosine kinase and adenosine deaminase were inhibited by 5-iodotubercidin and deoxycoformycin respectively, was about 18 nmol/min per g of cells in normoxia; it increased about 2-fold in anoxia, although AMP increased 8-16-fold in this condition. From studies with inhibitors of membrane 5'-nucleotidase and of S-adenosylhomocysteine hydrolase, it was deduced that adenosine is produced by the latter enzyme and by cytosolic 5'-nucleotidase in normoxia, and by cytosolic and membrane 5'-nucleotidases in anoxia. Unlike in normoxic hepatocytes, inhibition of adenosine kinase by 5-iodotubercidin neither elevated the adenosine concentration nor enhanced total purine release from adenine nucleotides in cells treated with N2/CO2 or KCN; it had only a slight effect in cells treated with amytal. This indicates that recycling of adenosine is suppressed or profoundly inhibited in anoxia. The rate of accumulation of adenosine in anoxia was several-fold lower than the rate of its rephosphorylation upon reoxygenation. It is concluded that the elevation of adenosine in anoxic hepatocytes is much more dependent on decreased recycling of adenosine by adenosine kinase than on increased production by dephosphorylation of AMP.

5'-Nucleotidase↗
PubMed1993

Residual adenylosuccinase activities in fibroblasts of adenylosuccinase-deficient children: parallel deficiency with adenylosuccinate and succinyl-AICAR in profoundly retarded patients and non-parallel deficiency in a mildly retarded girl.

Adenylosuccinase (ASase) catalyses both the conversion of succinyl-aminoimidazole carboxamide ribotide (succinyl-AICAR) into AICAR and that of adenylosuccinate into AMP in the synthesis of purine nucleotides. Its deficiency results in the accumulation in body fluids of the nucleosides corresponding to both substrates, succinyl-AICAriboside and succinyladenosine. Two main subtypes of the defect are type I with severe mental retardation and succinyladenosine/succinyl-AICAriboside ratios around 1, and type II with slight mental delay and succinyladenosine/succinyl-AICAriboside ratios around 4. We report that in fibroblasts of type I patients, the activity of ASase with both adenylosuccinate and succinyl-AICAR is about 30% of normal. In contrast, in type II fibroblasts, the activity with adenylosuccinate is only 3% of normal, whereas that with succinyl-AICAR is also 30% of normal. If also present in other tissues, this non-parallel deficiency provides an explanation for the higher concentration of succinyladenosine in type II. In type I fibroblasts, ASase is further characterized mainly by a 3-fold to 4-fold increase in Km for succinyl-AICAR, and by retarded elution from an anion exchanger. In type II fibroblasts, ASase is characterized by a similar increase in Km for succinyl-AICAR but by a potent inhibition by KCl and nucleoside triphosphates, and by a normal elution profile. These results suggest a modification of the surface charge of ASase in type I, and the addition of one or more positively charged residues in the active site in type II.

Adenosine Monophosphate↗
PubMed1993

Functional studies in fibroblasts of adenylosuccinase-deficient children.

In fibroblasts of severely retarded (type I) adenylosuccinase (ASase)-deficient children, activities with the two substrates of the enzyme, succinylaminoimidazole carboxamide ribotide (succinyl-AICAR) and adenylosuccinate are decreased in parallel, to about 30% of normal. In a markedly less retarded (type II) patient, ASase activity with adenylosuccinate reaches only 3% of normal, whereas activity with succinyl-AICAR is also about 30% of normal. To assess the functional significance of a partial versus a profound deficiency of ASase, precursor incorporation studies were performed in intact fibroblasts. In cells from controls and from type I patients, incorporation of 0.2 mmol/L [14C]formate into adenine and guanine nucleotides was not accompanied by accumulation of either [14C]succinyl-AICAR or [14C]adenylosuccinate. Similarly, incorporation of 20 mumol/L [14C]hypoxanthine was not accompanied by accumulation of [14C]adenylosuccinate. In contrast, in fibroblasts of the type II patient, in accordance with the profound deficiency of ASase with adenylosuccinate, and with the inhibitory effect of Cl- and nucleotides on the activity with succinyl-AICAR, incorporation of [14C]formate resulted in accumulation of [14C]succinyl-AICAR and [14C]adenylosuccinate, and incorporation of [14C]hypoxanthine in a marked build-up of [14C]adenylosuccinate. That both precursors were still incorporated into the adenine nucleotides of the fibroblasts of the type II patient indicates that adenylate synthesis remains possible even with 3% residual ASase activity, as also shown by their grossly normal ATP concentrations. The results suggest that the pathophysiology of ASase deficiency may be mediated at least in part by accumulation of succinyladenosine and succinyl-AICAriboside.

Adenosine Monophosphate↗
PubMed1992

Inhibition of glycolysis by 5-amino-4-imidazolecarboxamide riboside in isolated rat hepatocytes.

5-Amino-4-imidazolecarboxamide riboside (AICAriboside; Z-riboside), the nucleotide corresponding to AICAribotide (AICAR or ZMP), an intermediate of the 'de novo' pathway of purine nucleotide biosynthesis, has been shown to inhibit gluconeogenesis in isolated rat hepatocytes [Vincent, Marangos, Gruber & Van den Berghe (1991) Diabetes 40, 1259-1266]. We now report that glycosis is also inhibited and even more sensitive to AICAriboside in these cells. In hepatocyte suspensions from fasted rats, production of lactate from 15 mM-glucose was half-maximally inhibited by 25-50 microM-AICAriboside. AICAriboside influenced two regulatory steps of glycolysis: (1) it decreased the release of 3H2O from [2-3H]glucose and the concentrations of both glucose 6-phosphate and fructose 6-phosphate, indicating that it diminished the phosphorylation of glucose by glucokinase; (2) it decreased the concentration of fructose 2,6-bisphosphate (Fru-2,6-P2), the main physiological stimulator of liver 6-phosphofructo-1-kinase. Further studies showed that AICAriboside induced an inactivation of 6-phosphofructo-2-kinase, the enzyme that produces Fru-2,6-P2, without affecting the concentration of cyclic AMP. Similarly to the inhibiton of gluconeogenesis by AICAriboside, the inhibition of glycolysis became apparent after an approx. 10 min latency and persisted when the cells were washed after addition of AICAriboside, strongly suggesting that the effects were also exerted by the Z-nucleotides, which accumulate after addition of AICAriboside to hepatocytes. An increased uptake of lactate was evident when 50-200 microM-AICAriboside was added 15 min after addition of glucose. This can be explained by the higher sensitivity of glycolysis, as compared with gluconeogenesis, to inhibition by AICAriboside, and reveals the simultaneous operation of both processes.

Aminoimidazole Carboxamide↗
PubMed1992

The purine nucleotide cycle and its molecular defects.

Three enzymes of purine metabolism, adenylosuccinate synthetase, adenylosuccinate lyase and AMP deaminase, have been proposed to form a functional unit, termed the purine nucleotide cycle. This cycle converts AMP into IMP and reconverts IMP into AMP via adenylosuccinate, thereby producing NH3 and forming fumarate from aspartate. In muscle, the purine nucleotide cycle has been shown to function during intense exercise; the metabolic flux through the cycle has been proposed to play a role in the regeneration of ATP by pulling the adenylate kinase reaction in the direction of formation of ATP, and by providing Krebs cycle intermediates. In kidney, the purine nucleotide cycle was shown to account for the release of NH3 under the normal acid-base status, but not under acidotic conditions. In brain, the purine nucleotide cycle might function under conditions that induce a loss of ATP, and thereby contribute to its recovery. There is no evidence that the purine nucleotide cycle operates in liver. Deficiency of muscle AMP deaminase is an apparently frequent disorder, which might affect approximately 2% of the general population. The observation that it can be found in clinically asymptomatic individuals suggests, paradoxically, that the ATP-regenerating function which has been attributed to the purine nucleotide cycle is not essential for muscle function. Further work should be aimed at identifying the conditions under which AMP deaminase deficiency becomes symptomatic. Adenylosuccinate lyase deficiency provokes psychomotor retardation, often accompanied by autistic features. Its clinical heterogeneity justifies systematic screening in patients with unexplained mental deficiency. Additional studies are required to determine the mechanisms whereby this enzyme defect results in psychomotor retardation.

Animals↗
PubMed1992

ATP activates P2x-contracting and P2y-relaxing purinoceptors in the smooth muscle of mouse vas deferens.

1. The mechanism for the low potency of exogenous ATP in producing contraction at the P2x-purinoceptors in the smooth muscle of the mouse vas deferens (VD) was examined. 2. The measure of the breakdown of ATP in contact with the VD showed that its degradation was limited and did not account for its weak contractile effect. 3. Externally applied, ATP induced a small and transient contraction but a marked and prolonged increase of the cytosolic Ca2+ concentration ([Ca2+]i), which suggests an efficient binding to the P2x-purinoceptors. Such a calcium-force dissociation was not observed with beta, gamma-methylene ATP (beta, gamma-Me-ATP), a structural ATP analogue. 4. The force response of precontracted VD to ATP was biphasic, consisting of a small initial contraction followed by a sustained marked relaxation. In contrast, beta, gamma-Me-ATP elicited a pronounced contraction without ensuing relaxation. 5. ATP was more potent than adenosine in producing relaxation, and the relaxation was not antagonized by 8-phenyltheophylline, suggesting the activation of P2-purinoceptors. 6. For this relaxation, the rank order of potency was 2-methyl-thio-ATP (2-MeSATP) > ATP > beta, gamma-Me-ATP, which is characteristic for the P2y-purinoceptors. 7. Reactive Blue 2, a P2y-purinoceptor antagonist, was found to reduce the relaxation mediated by ATP. 8. These results indicate that ATP acts in VD not only on contracting but also on relaxing P2-purinoceptors, eliciting thereby overlapping opposite effects. In VD, the classical low potency of ATP or contraction is thus not explained by its low bioavailability or its low binding, but rather by its low specificity for the contracting P2x-purinoceptors, leading to the activation of the relaxing P2y-purinoceptors.

Adenosine Diphosphate↗
PubMed1991

Radiochemical assay of adenylosuccinase: demonstration of parallel loss of activity toward both adenylosuccinate and succinylaminoimidazole carboxamide ribotide in liver of patients with the enzyme defect.

A radiochemical assay for adenylosuccinase, an enzyme which intervenes twice in the biosynthesis of adenine nucleotides, has been developed. The two substrates of the enzyme, succinylaminoimidazole carboxamide ribotide (SAICAR) and adenylosuccinate (S-AMP), were synthesized in radioactive form by incubating [2,3-14C]fumarate and, respectively, AICAR and AMP with partially purified adenylosuccinase from yeast. Enzyme activities were determined by measuring the release of labeled fumarate after its separation from the substrate by chromatography on polyethyleneimine thin-layer plates. The ratio of the activity of adenylosuccinase measured with SAICAR compared to that with S-AMP was about 1 in crude extracts of rat liver and muscle and around 0.5 in human liver. In rat and human liver, but not in rat muscle, 20 to 40% of both activities of adenylosuccinase were lost after freezing at -80 degrees C followed by thawing. In the liver of patients with adenylosuccinase deficiency, in whom the deficiency had hitherto been measured only with S-AMP, the activity of the enzyme toward S-AMP and SAICAR was found to be lost in parallel. This is in accordance with the finding that both SAICA-riboside and succinyladenosine accumulate in adenylosuccinase-deficient patients.

Adenosine Monophosphate↗