Low Levels Of ADP Research Articles

Metabolic fate of glucose and candidate signaling and excess-fuel detoxification pathways in pancreatic β-cells

Glucose metabolism promotes insulin secretion in β-cells via metabolic coupling factors that are incompletely defined. Moreover, chronically elevated glucose causes β-cell dysfunction, but little is known about how cells handle excess fuels to avoid toxicity. Here we sought to determine which among the candidate pathways and coupling factors best correlates with glucose-stimulated insulin secretion (GSIS), define the fate of glucose in the β-cell, and identify pathways possibly involved in excess-fuel detoxification. We exposed isolated rat islets for 1 h to increasing glucose concentrations and measured various pathways and metabolites. Glucose oxidation, oxygen consumption, and ATP production correlated well with GSIS and saturated at 16 mm glucose. However, glucose utilization, glycerol release, triglyceride and glycogen contents, free fatty acid (FFA) content and release, and cholesterol and cholesterol esters increased linearly up to 25 mm glucose. Besides being oxidized, glucose was mainly metabolized via glycerol production and release and lipid synthesis (particularly FFA, triglycerides, and cholesterol), whereas glycogen production was comparatively low. Using targeted metabolomics in INS-1(832/13) cells, we found that several metabolites correlated well with GSIS, in particular some Krebs cycle intermediates, malonyl-CoA, and lower ADP levels. Glucose dose-dependently increased the dihydroxyacetone phosphate/glycerol 3-phosphate ratio in INS-1(832/13) cells, indicating a more oxidized state of NAD in the cytosol upon glucose stimulation. Overall, the data support a role for accelerated oxidative mitochondrial metabolism, anaplerosis, and malonyl-CoA/lipid signaling in β-cell metabolic signaling and suggest that a decrease in ADP levels is important in GSIS. The results also suggest that excess-fuel detoxification pathways in β-cells possibly comprise glycerol and FFA formation and release extracellularly and the diversion of glucose carbons to triglycerides and cholesterol esters.

Differential interaction of hGDH1 and hGDH2 with manganese: Implications for metabolism and toxicity

Manganese (Mn) is an essential trace element that serves as co-factor for many important mammalian enzymes. In humans, the importance of this cation is highlighted by the fact that low levels of Mn cause developmental and metabolic abnormalities and, on the other hand, chronic exposure to excessive amounts of Mn is characterized by neurotoxicity, possibly mediated by perturbation of astrocytic mitochondrial energy metabolism. Here we sought to study the effect of Mn on the two human glutamate dehydrogenases (hGDH1 and hGDH2, respectively), key mitochondrial enzymes involved in numerous cellular processes, including mitochondrial metabolism, glutamate homeostasis and neurotransmission, and cell signaling. Our studies showed that, compared to magnesium (Mg) and calcium (Ca), Mn exerted a significant inhibitory effect on both human isoenzymes with hGDH2 being more sensitive than hGDH1, especially under conditions of low ADP levels. Specifically, in the presence of 0.25 mM ADP, the Mn IC50 was 1.14 ± 0.02 mM and 1.54 ± 0.08 mM for hGDH2 and for hGDH1, respectively (p = 0.0001). Increasing Mn levels potentiated this differential effect, with 3 mM Mn inhibiting hGDH2 by 96.5% and hGDH1 by 70.2%. At 1 mM ADP, the Mn IC50 was 1.84 ± 0.02 mM and 2.04 ± 0.07 mM (p = 0.01) for hGDH2 and hGDH1, respectively, with 3 mM Mn inhibiting hGDH2 by 93.6% and hGDH1 by 70.9%. These results were due to the sigmoidal inhibitory curve of Mn that was more pronounced for hGDH2 than for hGDH1. Indeed, at 0.25 mM, the Hill coefficient value was higher for hGDH2 (3.42 ± 0.20) than for hGDH1 (1.94 ± 0.25; p = 0.0002) indicating that interaction of Mn with hGDH2 was substantially more co-operative than for hGDH1. These findings, showing an enhanced sensitivity of the hGDH2 isoenzyme to Mn, especially at low ADP levels, might be of pathophysiological relevance under conditions of Mn neurotoxicity.

Is adiponectin a marker of preclinical atherosclerosis in kidney transplantation?

The aim of this study was to analyze the relationship between pre-transplant adiponectin (pre-ADP), abnormalities in glucose homeostasis (AGH) at three months post-transplantation, and preclinical atherosclerosis in non-diabetic patients prior to kidney transplantation (KT). We carried out a multicenter study in 157 non-diabetic KT patients (66.5% men; age: 50±13 yr). Pre-ADP levels were analyzed using radioimmunoassay. Carotid ultrasound was performed to determine carotid intima-media thickness (c-IMT). Oral glucose tolerance test was carried out to classify patients according ADA criteria. Of the patients, 52.8% had AGH. Median pre-ADP was 19.5 (14-27) μg/mL. An inverse correlation was found between ADP and HOMA index (r=-0.432; p<0.001). Median c-IMT was 0.6 (0.48-0.71) mm. Significant inverse correlation existed between ADP and c-IMT on both sides (p<0.05). Patients with c-IMT >0.6 mm had more AGH (p=0.012) and lower ADP levels (p=0.02). We performed a logistic regression analysis using preclinical atherosclerosis (c-IMT ≥0.6 mm) as dependent variable and sex, age, BMI, ADP, AGH, and HOMA index as independent variables of altered c-IMT. Age, pre-ADP, and AGH were independent risk factors for elevated c-IMT. Patients with AGH have a greater presence of preclinical atherosclerosis. ADP has an inverse relationship with AGH and is an independent marker of preclinical atherosclerosis.

Abstract 5008: Effect of Clopidogrel Discontinuation on Platelet Function

Objective: Clopidogrel discontinuation is associated with increased incidence of ischemic events, which may signal a pharmacological rebound. We evaluated the impact of clopidogrel discontinuation on platelet function in CAD patients. Methods: Platelet aggregation was measured by VerifyNow ® P2Y 12 and by light transmission aggregometry (LTA) in response to ADP 1, 2, 3, 5, 10, and 20 μ M, in 37 CAD patients scheduled to discontinue clopidogrel treatment (aspirin treated patients clinically stable 1 year after clopidogrel initiation), and 37 CAD patients not taking clopidogrel. Platelet function was assessed the day before clopidogrel cessation and 1, 3, 7, 14, 21, 28 days after. Results: Subjects were on average 65±10 years old, and presented classic CAD risk factors similarly treated between groups. Subjects discontinuing clopidogrel were more likely to have undergone stenting. VerifyNow ® P2Y 12 did not detect any rebound platelet activity, but ADP-induced LTA showed significant increase in platelet aggregation, particularly at low ADP levels, suggestive of platelet hypersensitivity (Fig ). Rebound platelet activity was detectable at 7 days after clopidogrel cessation, and remained higher than in controls 28 days of discontinuation. No clinical events were seen at 28 days in either group. Conclusion: Platelet sensitization to ADP as a consequence of chronic clopidogrel administration may partially explain the recrudescence of ischemic events following clopidogrel discontinuation in otherwise stable CAD patients. Further research into the pathophysiological processes of platelet function recovery is required. Figure: Platelet aggregation as a function of time

Shiga Toxin 2 and Lipopolysaccharide Induce Human Microvascular Endothelial Cells To Release Chemokines and Factors That Stimulate Platelet Function

Shiga toxins (Stxs) produced by Shigella dysenteriae type 1 and enterohemorrhagic Escherichia coli are the most common cause of hemolytic-uremic syndrome (HUS). It is well established that vascular endothelial cells, mainly those located in the renal microvasculature, are targets for Stxs. The aim of the present research was to evaluate whether E. coli-derived Shiga toxin 2 (Stx2) incubated with human microvascular endothelial cells (HMEC-1) induces release of chemokines and other factors that might stimulate platelet function. HMEC-1 were exposed for 24 h in vitro to Stx2, lipopolysaccharide (LPS), or the Stx2-LPS combination, and chemokine production was assessed by immunoassay. More interleukin-8 was released than stromal cell-derived factor 1alpha (SDF-1alpha) or SDF-1beta and RANTES. The Stx2-LPS combination potentiated chemokine release, but Stx2 alone caused more release of SDF-1alpha at 24 h than LPS or Stx2-LPS did. In the presence of low ADP levels, HMEC-1 supernatants activated platelet function assessed by classical aggregometry, single-particle counting, granule secretion, P-selectin exposure, and the formation of platelet-monocyte aggregates. Supernatants from HMEC-1 exposed only to Stx2 exhibited enhanced exposure of platelet P-selectin and platelet-THP-1 cell interactions. Blockade of platelet cyclooxygenase by indomethacin prevented functional activation. The chemokine RANTES enhanced platelet aggregation induced by SDF-1alpha, macrophage-derived chemokine, or thymus and activation-regulated chemokine in the presence of very low ADP levels. These data support the hypothesis that microvascular endothelial cells exposed to E. coli O157:H7-derived Stx2 and LPS release chemokines and other factors, which when combined with low levels of primary agonists, such as ADP, cause platelet activation and promote the renal thrombosis associated with HUS.

ADP receptor antagonists inhibit platelet aggregation induced by the chemokines SDF-1, MDC and TARC

The ability of the chemokines SDF-1, MDC and TARC to induce platelet aggregation depends strongly on low levels of ADP. The ADP receptors involved have now been characterized using the P2Y 1 and P2T AC receptor antagonists, A2P5P and AR-C69931MX. Stimulation of aggregation by the chemokines at 10 s was not blocked by AR-C69931MX, but was strongly inhibited by A2P5P. Pertussis toxin abolished the chemokine-stimulated aggregation. We conclude that the P2Y 1 ADP receptor plays a critical role in the initial phases of SDF-1-, MDC- and TARC-induced platelet aggregation, which involve a pertussis toxin-sensitive G protein.

Nerve tissue-specific human glutamate dehydrogenase that is thermolabile and highly regulated by ADP.

Glutamate dehydrogenase (GDH), an enzyme that is central to the metabolism of glutamate, is present at high levels in the mammalian brain. Studies on human leukocytes and rat brain suggested the presence of two GDH activities differing in thermal stability and allosteric regulation, but molecular biological investigations led to the cloning of two human GDH-specific genes encoding highly homologous polypeptides. The first gene, designated GLUD1, is expressed in all tissues (housekeeping GDH), whereas the second gene, designated GLUD2, is expressed specifically in neural and testicular tissues. In this study, we obtained both GDH isoenzymes in pure form by expressing a GLUD1 cDNA and a GLUD2 cDNA in Sf9 cells and studied their properties. The enzymes generated showed comparable catalytic properties when fully activated by 1 mM ADP. However, in the absence of ADP, the nerve tissue-specific GDH showed only 5% of its maximal activity, compared with approximately 40% showed by the housekeeping enzyme. Low physiological levels of ADP (0.05-0.25 mM) induced a concentration-dependent enhancement of enzyme activity that was proportionally greater for the nerve tissue GDH (by 550-1,300%) than of the housekeeping enzyme (by 120-150%). Magnesium chloride (1-2 mM) inhibited the nonactivated housekeeping GDH (by 45-64%); this inhibition was reversed almost completely by ADP. In contrast, Mg2+ did not affect the nonstimulated nerve tissue-specific GDH, although the cation prevented much of the allosteric activation of the enzyme at low ADP levels (0.05-0.25 mM). Heat-inactivation experiments revealed that the half-life of the housekeeping and nerve tissue-specific GDH was 3.5 and 0.5 h, respectively. Hence, the nerve tissue-specific GDH is relatively thermolabile and has evolved into a highly regulated enzyme. These allosteric properties may be of importance for regulating brain glutamate fluxes in vivo under changing energy demands.

Mechanism of Inhibitory Action on Platelet Activation of a Phospholipase A2 Isolated from Lachesis muta (Bushmaster) Snake Venom

Crude venom from Lachesis muta exhibited procoagulant, proteolytic and phospholipase A2 activities. A phospholipase A2, denoted LM-PLA2 was purified from L. muta venom to homogeneity, through a combination of chromatographic steps involving gel-filtration on Sephacryl S-200 HR and reverse phase chromatography on a C2/C18 column. LM-PLA2 presented a single polypeptide chain with an isoelectric point at pH 4.7 and apparent molecular weight of 17 kDa. Partial aminoacid sequence indicated a high degree of homology for LM-PLA2 with other PLA2 from different sources. LM-PLA2 displayed a potent enzymatic activity as measured by indirect hemolysis of red blood cells but it was neither lethal when injected i.p. into mice nor did it present anticoagulant activity. Furthermore, LM-PLA2 displayed a moderate inhibitory activity on the aggregation of rabbit platelets induced by low levels of ADP, thrombin and arachidonate. In contrast, platelet aggregation induced by high doses of collagen was strongly inhibited by LM-PLA2 as well as ATP-release. Treatment of the protein with p-bromophenacyl bromide or 2-mercaptoethanol, as well as thermal inactivation studies, suggested that the platelet inhibitory effect of LM-PLA2 is dependent on its enzymatic activity. Thus, the platelet inhibitory activity of LM-PLA2 was shown to be dependent on the hydrolysis of plasma phospholipids and/or lipoproteins, most probably those rich in phosphatidylcholine. Surprisingly, lysophosphatidylcholine released by LM-PLA2 from plasma was shown to preferentially inhibited collagen-induced platelet aggregation, in contrast to other PLA2s, whose plasma hydrolytic products indistinctly affect platelet's response to several agonists.

Energization of the membrane prevents the formation of tight inactive complexes of ATPase with MgADP in submitochondrial particles

Mitochondrial ATPase binds ADP tightly, forming inactive complexes. Dissociation of the complexes is blocked by azide. Azide inhibits neither ATP hydrolysis at high δ μ H + nor ATP-dependent NAD + reduction, provided that the ADP concentration is higher than 10 −4 M. At lower ADP levels, azide is inhibitory. These data suggest that in the presence of ATP δ μ H + prevents the fromation of one of the inactive complexes. In the absence of ATP at high δ μ H +, azide-sensitive complexes are not formed at any ADP concentrations tested (5 × 10 −7-5 × 10 −4 M). The inactive E·ADP complexes can play a significant role in the regulation of ATPase in mitochondria, preventing futile ATP hydrolysis at low δ μ H +.

The Coupling of Electron Flow to ATP Synthesis in Pea and Maize Mesophyll Chloroplasts

The rate of nonphosphorylating electron transport (in the absence of ADP and inorganic phosphate) in well-coupled (ATP/2e(-) = 0.9-1.1) maize mesophyll chloroplasts is not modulated by external pH (6.5-8.5), low levels of ADP or ATP, or energy transfer inhibitors, e.g. triphenyltin and Hg(2+) ions. In contrast nonphosphorylating electron flow in pea chloroplasts is sensitive to alterations in medium pH, and to the presence of adenine nucleotides and energy transfer inhibitors in the assay medium. Although ATP is without effect on the rate of basal electron transport in maize chloroplasts, steady-state proton uptake is stimulated 3- to 5-fold by low levels of ATP. These results suggest that differences may exist in the manner in which the coupling factor complex controls proton efflux from the intrathylakoid space in C(3) and C(4) mesophyll chloroplasts.

Proton efflux through the chloroplast ATP synthase (CF0 · CF1) in the presence of sulfhydryl-modifying agents

The rate of photosynthetic electron transport measured in the absence of ADP and Pi is stimulated by low levels of Hg2+ or Ag+ (50% stimulation ≈ 3 Hg2+ or 6 Ag+/100 chlorophyll) to a plateau equal to the transport rate under normal phosphorylating conditions (i.e. +ADP, +Pi). Chloroplasts pretreated in the light under energizing conditions with N-ethylmaleimide show a similar stimulation of non-phosphorylating electron transport. The stimulations of non-phosphorylating electron transport by Hg2+, Ag+ and N-ethylmaleimide are reversed by the CF1 inhibitor phlorizin, the CF0 inhibitor triphenyltin chloride, and can be further stimulated by uncouplers such as methylamine. The Hg2+ and N-ethylmaleimide stimulations, but not the Ag+ stimulation, are completely reversed by low levels of ADP (2 μM), ATP (2 μM), and Pi (400 μM). Ag+, which is a potent inhibitor of ATP synthesis, has little or no effect upon phosphorylating electron transport (+ADP, +Pi). Concomitant with the stimulations of non-phosphorylating electron transport by Hg2+, Ag+ and ADP + Pi, there is a decrease in the level of membrane energization (as measured by atebrin fluorescence quenching) which is reversed when the CF0 channel is blocked by triphenyltin. These results suggest that modification of critical CF1 sulfhydryl residues by Hg2+, Ag+ or N-ethylmaleimide leads to the loss of intra-enzyme coupling between the transmembrane protontransferring and the ATP synthesis activities of the CF0-CF1 ATP synthase complex.

Pyruvate kinase from the liver and kidney of Arapaima gigas

Pyruvate kinases from the kidney and liver of the osteoglossid Arapaima gigas were partially purified and characterized kinetically. The two enzymes have different elect rophoretic mobilities at pH 7.0, and while they share some qualitative similarities they show quantitative differences in their catalytic and regulatory properties. Both enzymes are activated by fructose 1.6-bisphosphate and inhibited by low levels of alanine and MgATP. The liver isozyme shows hyperbolic phosphoenolpyruvate binding, with a K1 for alanine inhibition of 0.7 mM and a K1 for MgATP inhibition of 1.0 mM. In contrast, the kidney isozyme shows cooperative phosphoenolpyruvate binding, which is accentuated at low levels of ADP. MgATP inhibition does not increase the cooperativity and shows an apparent K1 of 1.68 mM. The inhibition of alanine leads to considerable increases in the cooperativity and is effective at 1 mM and lower levels. Fructose 1.6-bisphosphate completely reverses the inhibition by alanine for both isozymes, while only leading to a partial reversal of the MgATP inhibition. These regulatory properties of both the kidney and the liver isozymes suit them for function in tissues which undergo both glycolysis and gluconeogenesis.

Control of ammoniagenesis in the kidney of water- and air-breathing osteoglossids: characterization of glutamate dehydrogenase

Glutamate dehydrogenases (EC 1.4.1.2) from the kidney of Osteoglossum bicirrhosum (called aruana) and Arapaima gigas were kinetically characterized. The two enzymes exhibited several common characteristics including Vmax activity ratio, pH optimum, affinity for cofactors, a marked preference for NAD(H) over NADP(H), and a very low affinity for NH4+. A variety of regulatory metabolites affected both enzymes. GTP and GDP were inhibitory while ADP, ATP, AMP, and leucine activated the enzymes. Both enzymes displayed potent product inhibition which was partially reversed by low levels of ADP. Arapaima kidney glutamate dehydrogenase was tightly regulated by the adenylate and guanylate nucleotides, inhibition by GTP and GDP and deinhibition by ADP and AMP being much stronger for this enzyme than for the aruana enzyme. Aruana glutamate dehydrogenase, however, was more responsive to NAD–NADH control. The enzyme was more sensitive to NAD(H) product inhibition and this inhibition was poorly reversed by ADP. From these data, it was concluded that both fish kidney glutamate dehydrogenases could function in glutamate oxidation in vivo. However, the Arapaima enzyme appeared most clearly adapted to a catabolic role, activity being more tightly linked to the energy status of the mitochondrion. Conversely, the aruana enzyme displayed regulatory properties allowing it the potential to function in NADH oxidation during periods of hypoxic stress.

On the mechanism of carbamoylphosphate synthetase.

Studies were undertaken to investigate whether 2‐acetamido glutaric anhydride is an intermediate in the synthesis of carbamoylphosphate. Experiments were carried out with pools of 2‐acetamido‐glutaric anhydride, its hydroxamate and NH2OH to trap the anhydride. The anhydride was also tested as an inhibitor. The experiments show no evidence for the participation of free 2‐acetamido‐glutaric anhydride as an intermediate.The effect of NH2OH on the synthesis of ATP from carbamoylphosphate and ADP was investigated in order to clarify whether this reaction is an integral part of the overall reaction catalyzed by carbamoylphosphate synthetase and whether there is a common intermediate which could react with NH2OH. The unexpected observation was made that NH2OH is an inhibitor of ATP synthesis at low levels of ADP, and that the enzyme has a high affinity for ADP. These results together with other experiments on carbamoylphosphate binding by the enzyme point to a competition between ADP site and NH2OH. No increase in the release of Pi from carbamoylphosphate in the presence of both acetylglutamate and NH2OH was observed; therefore the intermediate responsible for the acetylglutamate NH2OH‐dependent ATPase is not formed extensively from carbamoylphosphate.The activation of carbamoylphosphate synthetase by acetylglutamate for both the forward and back reactions was studied. The enzyme was activated with acetylglutamate and rapidly separated from the cofactor on Sephadex. There was no evidence for retention of activation, indicating a fast return to the non‐activated state. Therefore, enzyme samples were activated, diluted and assayed immediately and after time intervals. A time‐dependent reversibility of the activated enzyme for both the forward and back reactions is illustrated.

Ultrastructural studies of beef heart mitochondria II. Adenine nucleotide induced modifications of mitochondrial morphology

Low levels of ADP, dADP, ATP and dATP induce a distinctive ultrastructure in the network of inner mitochondrial membranes whilst oxygen consumption is essentially unchanged. Induction of a coalesced membrane morphology by these adenine nucleotides is not prevented by oligomycin or low temperature (0° C), but is blocked by atractyloside. Other purine and pyrimidine nucleotides will not substitute for adenine nucleotides. These observations strongly indicate that gross morphological transitions of the inner membranes induced by adenine nucleotides are not intermediate energy transducing forms of oxidative phosphorylation, but probably reflect the consequences of specific and stoichiometric binding of adenine nucleotides to the translocation sites of the inner mitochondrial membrane.

Regulation of pyruvate kinase

The mechanism and sequence of events of the induction of liver pyruvate kinase by sucrose and fructose administration were examined. Actinomycin and cycloheximide prevented the sucrose and fructose induced rise in pyruvate kinase activity. In addition to ADP, GDP, UDP, IDP and, to a minor extent, CDP and TTP were able to function as acceptors for pyruvate kinase for both liver and hepatoma. At saturating ADP levels ATP, GTP, UTP, ITP, and, to a minor extent, CTP were inhibitors of liver pyruvate kinase activity. GTP was a stronger inhibitor for hepatoma pyruvate kinase whereas UTP was not inhibitory. At low ADP levels TTP was a good inhibitor and the other nucleotides are being studied. l-Alanine was a strong competitive inhibitor of liver pyruvate kinase and of the hepatoma enzyme; however, it had little effect on the muscle or brain enzyme. In contrast, l-phenylalanine was a good inhibitor of rat and human brain pyruvate kinase. It is suggested that the inhibition of human adult and fetal brain pyruvate kinase by l-phenylalanine may have a role in the metabolic damage in phenylketonuria. The chief mechanisms governing the rate and direction of the opposing metabolic pathways of gluconeogenesis and glycolysis in the liver were discussed.