inosinic-acid and adenylosuccinate

Pancreatic islet failure, involving loss of glucose-stimulated insulin secretion (GSIS) from islet β cells, heralds the onset of type 2 diabetes (T2D). To search for mediators of GSIS, we performed metabolomics profiling of the insulinoma cell line 832/13 and uncovered significant glucose-induced changes in purine pathway intermediates, including a decrease in inosine monophosphate (IMP) and an increase in adenylosuccinate (S-AMP), suggesting a regulatory role for the enzyme that links the two metabolites, adenylosuccinate synthase (ADSS). Inhibition of ADSS or a more proximal enzyme in the S-AMP biosynthesis pathway, adenylosuccinate lyase, lowers S-AMP levels and impairs GSIS. Addition of S-AMP to the interior of patch-clamped human β cells amplifies exocytosis, an effect dependent upon expression of sentrin/SUMO-specific protease 1 (SENP1). S-AMP also overcomes the defect in glucose-induced exocytosis in β cells from a human donor with T2D. S-AMP is, thus, an insulin secretagogue capable of reversing β cell dysfunction in T2D.

Topics: Adenosine Monophosphate; Adenylosuccinate Lyase; Adenylosuccinate Synthase; Animals; Cell Line, Tumor; Cysteine Endopeptidases; Diabetes Mellitus, Type 2; Endopeptidases; Enzyme Inhibitors; Exocytosis; Gene Expression Regulation; Glucose; Guanine; Humans; Inosine Monophosphate; Insulin; Insulin Secretion; Insulin-Secreting Cells; Metabolome; Mycophenolic Acid; Patch-Clamp Techniques; Primary Cell Culture; Rats; Rats, Sprague-Dawley; Signal Transduction

Dissociation constants of Escherichia coli adenylosuccinate synthetase with IMP, GTP, adenylosuccinate, and AMP (a competitive inhibitor for IMP) were determined by measuring the extent of quenching of the intrinsic tryptophan fluorescence of the enzyme. The enzyme has one binding site for each of these ligands. Aspartate and GDP did not quench the fluorescence to any great extent, and their dissociation constants could not be determined. These ligand binding studies were generally supportive of the kinetic mechanism proposed earlier for the enzyme. Cys291 was modified with the fluorescent chromophores N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulfonate and tetramethylrhodamine maleimide in order to measure enzyme conformational changes attending ligand binding. The excitation and emission spectra of these fluorophores are not altered by the addition of active site binding ligands. TbGTP and TbGDP were used as native reporter groups, and changes in their fluorescence on complexing with the enzyme and various ligands made it possible to detect conformational changes occurring at the active site. Evidence is presented for abortive complexes of the type: enzyme-TbGTP-adenylosuccinate and enzyme-TbGTP-adenylosuccinate-aspartate. These results suggest that the IMP and aspartate binding sites are spatially separated.

Topics: Adenosine Monophosphate; Adenylosuccinate Synthase; Binding Sites; Cysteine; Escherichia coli; Fluorescent Dyes; Guanosine Triphosphate; Inosine Monophosphate; Kinetics; Ligands; Naphthalenesulfonates; Protein Conformation; Rhodamines; Spectrometry, Fluorescence

Nucleotide metabolism was studied in rats during and following the induction of 10 min of forebrain ischemia (four-vessel occlusion model). Purine and pyrimidine nucleotides, nucleotides, and bases in forebrain extracts were quantitated by HPLC with an ultraviolet detector. Ischemia resulted in a severe reduction in the concentration of nucleoside triphosphates (ATP, GTP, UTP, and CTP) and an increase in the concentration of AMP, IMP, adenosine, inosine, hypoxanthine, and guanosine. During the recovery period, both the phosphocreatine level and adenylate energy charge were rapidly and completely restored to the normal range. ATP was only 78% of the control value at 180 min after ischemic reperfusion. Levels of nucleosides and bases were elevated during ischemia but decreased to values close to those of control animals following recirculation. Both the decrease in the adenine nucleotide pool and the incomplete ATP recovery were caused by insufficient reutilization of hypoxanthine via the purine salvage system. The content of cyclic AMP, which transiently accumulated during the early recirculation period, returned to the control level, paralleling the decrease of adenosine concentration, which suggested that adenylate cyclase activity during reperfusion is modulated by adenosine A2 receptors. The recovery of CTP was slow but greater than that of ATP, GTP, and UTP. The GTP/GDP ratio was higher than that of the control animals following recirculation.

Topics: Adenosine; Adenosine Monophosphate; Adenosine Triphosphate; Animals; Brain; Chromatography, High Pressure Liquid; Cytidine Triphosphate; Guanine; Guanosine Diphosphate; Guanosine Triphosphate; Hypoxanthine; Hypoxanthines; Inosine; Inosine Monophosphate; Ischemic Attack, Transient; Male; Nucleotides; Phosphocreatine; Rats; Rats, Inbred Strains; Uridine Triphosphate

Metabolites of 5-amino-4-imidazolecarboxamide riboside (Z-riboside) have potential roles in the regulation of cellular metabolism and as pharmacological agents in several pathological situations. Before studying Z-riboside metabolism it was necessary to develop methods for identifying and quantitating 5(4)-amino-4(5)-imidazolecarboxamide metabolites. These studies utilized Chinese hamster ovary fibroblast auxotrophic mutants to identify and isolate compounds relevant to Z-riboside metabolism by a combination of high performance liquid chromatographic procedures. In order to study Z-riboside metabolism wild-type and mutant cells were cultured in Z-riboside. This ribosyl precursor to a purine de novo intermediate does not undergo any detectable phosphorolysis but rather is phosphorylated by adenosine kinase in an unregulated manner. This results in the intracellular accumulation of 5-amino-4-imidazolecarboxamide ribotide (ZMP), the levels of which control flow from Z-riboside to the following metabolites: 1) IMP and other purine nucleotides, 2) 5-amino-4-imidazole-N-succinocarboxamide ribotide (sZMP), and 3) 5-amino-4-imidazolecarboxamide riboside 5'-triphosphate (ZTP). At low ZMP concentrations, the predominant metabolic fate is IMP. Initially, IMP enters the adenylate and guanylate pools, but subsequently is hydrolyzed to inosine and this phosphorolyzed to hypoxanthine. At intermediate ZMP concentrations there is net retrograde flux through the bifunctional enzyme adenylosuccinate AMP lyase resulting in sZMP synthesis and antegrade flux leads to the accumulation of adenylosuccinate. At high ZMP concentrations, ZTP accumulates. In addition to these effects on purine metabolism, pyrimidine nucleotide pools are depleted when ZMP accumulates. These results are discussed in relation to the regulation of purine nucleotide synthesis and the use of Z-riboside as a pharmacological intervention in pathophysiological situations.

Topics: Adenosine Monophosphate; Aminoimidazole Carboxamide; Animals; Cells, Cultured; Cricetinae; Cricetulus; Female; Fibroblasts; Imidazoles; Inosine Monophosphate; Ovary; Purine Nucleotides; Pyrimidine Nucleotides; Ribonucleosides; Ribonucleotides

The enzymes of the purine nucleotide cycle-AMP deaminase, adenylosuccinate synthetase, and adenylosuccinate lyase-were examined as a functional unit in an in vitro system which simulates the purine nucleotide composition of sarcoplasm. Activity of each cycle enzyme in extracts of rat skeletal muscle was observed to increase as ATP/ADP, reflecting the energy state of the system, was lowered from approximately 50 to 1. The increase in AMP deaminase activity could be attributed to effects of energy state and factors such as AMP concentration, which are obligatorily coupled to energy state. The increases in synthetase and lyase activities were accounted for by increases in the concentration of IMP and adenylosuccinate, respectively. The inhibitory influence of IMP concentration on synthetase activity reported in other systems was not observed in this system; synthetase activity progressively increased as IMP concentration was raised to approximately 4 mM, and apparent saturation occurred at concentrations above 4 mM. Also, adenylosuccinate was found to be an activator of AMP deaminase. The results of this study document that the activities of the enzymes of the purine nucleotide cycle increase in parallel at low energy states, and the components of the cycle function as a coordinated unit with individual enzyme activities linked via concentrations of cycle intermediates.

Topics: Adenosine Monophosphate; Adenylosuccinate Lyase; Adenylosuccinate Synthase; AMP Deaminase; Animals; Energy Metabolism; Enzyme Activation; In Vitro Techniques; Inosine Monophosphate; Kinetics; Male; Muscles; Purine Nucleotides; Rats; Rats, Inbred Strains

1. AMP, ADP, ATP, IMP, GDP, GTP and adenylosuccinate have been measured by high pressure liquid chromatography in three types of animal muscular dystrophy and in a human patient with Duchenne muscular dystrophy. 2. Abnormalities in nucleotide content varied from one dystrophy to another. 3. In each case, however, the ratio [total adenine nucleotide + IMP]/[total guanine nucleotides] was lower in dystrophic muscle, even when severely exercised or ischaemic muscles were used. 4. The practical advantages of this assay for diagnosis of muscular dystrophy are discussed.

Topics: Adenine Nucleotides; Adenosine Monophosphate; Animals; Chickens; Child, Preschool; Guanine Nucleotides; Humans; Inosine Monophosphate; Male; Mice; Mice, Inbred C57BL; Muscles; Muscular Dystrophies; Muscular Dystrophy, Animal; Purine Nucleotides

The following evidence demonstrates that ammonia production in muscle occurs via the reactions of the purine nucleotide cycle: i) Extracts of cytosol which lack glutamine dehydrogenase produce ammonia under conditions that mimic muscle doing work. In such extracts a member of the purine nucleotide cycle (AMP, IMP, or adenylosuccinate) must be present in order that ammonia production can take place, ii) Perfused hindleg of rat or hindleg in situ produces ammonia during exercise, and there is a concomitant production of IMP and adenylosuccinate. Exercise causes a decrease in the contents of glutamate, aspartate, and glutamine and an increase in the content of alanine of perfused hindleg of rat. However, output of alanine does not change or is diminished during exercise. Glutamine output is diminished by exercise. Epinephrine increases the output and tissue content of ammonia and glutamine. It decreases the output and content of alanine, and it decreases the contents of aspartate and glutamate.

Topics: Adenosine Diphosphate; Adenosine Monophosphate; Ammonia; Animals; Epinephrine; Inosine Monophosphate; Lactates; Muscles; Phosphocreatine; Physical Exertion; Purine Nucleotides; Rats; Succinates