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1.
Concentrations and rates of synthesis of phosphoribosylpyrophosphate (PP-Rib-P) and purine nucleotides were compared in fibroblasts cultured from 5 males with PP-Rib-P synthetase superactivity, 3 normal individuals, and 2 children with severe hypoxanthine-guanine phosphoribosyltransferase deficiency. Although all cell strains with PP-Rib-P synthetase superactivity showed increased PP-Rib-P concentration and generation, increased rates of PP-Rib-P-dependent purine synthetic pathways, and increased purine and pyrimidine nucleoside triphosphate concentrations, two subgroups were discernible. Three fibroblast strains with isolated catalytic defects in PP-Rib-P synthetase showed milder increases in PP-Rib-P concentration (2.5-fold normal) and generation (1.6- to 2.1-fold) and in rates of purine synthesis de novo (1.6- to 2.2-fold) and purine nucleoside triphosphate pools (1.5-fold) than did cells from 2 individuals with combined kinetic defects in PP-Rib-P synthetase, both with purine nucleotide inhibitor-resistance. Values for these processes in the latter two strains were, respectively, 5- to 6-fold, 2.6- to 3.2-fold, 4- to 7-fold, and 1.7- to 2.2-fold those of normal cells. In contrast to cells with catalytic defects, these cells also excreted an abnormally high proportion of labeled purines and resisted purine base-mediated inhibition of PP-Rib-P and purine nucleotide synthesis. Hypoxanthine-guanine phosphoribosyltransferase-deficient cells showed normal regulation of PP-Rib-P synthesis and normal nucleoside triphosphate pools despite increased rates of purine synthesis de novo and of purine excretion. Cells with PP-Rib-P synthetase superactivity thus synthesize purine nucleotides at increased rates as a consequence of increased PP-Rib-P production, despite increased purine nucleotide concentrations. These and additional findings provide evidence that regulation of purine synthesis de novo is effected at both the PP-Rib-P synthetase and amidophosphoribosyltransferase reactions.  相似文献   

2.
Human B lymphoblast lines severely deficient in hypoxanthine-guanine phosphoribosyltransferase (HGPRT) were selected for resistance to 6-thioguanine from cloned normal and phosphoribosylpyrophosphate (PP-Rib-P) synthetase-superactive cell lines and were compared with their respective parental cell lines with regard to growth and PP-Rib-P and purine nucleotide metabolism. During blockade of purine synthesis de novo with 6-methylthioinosine or aminopterin, inhibition of growth of all HGPRT-deficient cell lines was refractory to addition of Ade at concentrations which restored substantial growth to parental cell lines. Ade-resistant inhibition of growth of parental lines by 6-methylthioinosine, however, occurred during Ado deaminase inhibition. Insufficient generation of IMP (and ultimately guanylates) to support growth of lymphoblasts lacking HGPRT activity and blocked in purine synthesis de novo best explained these findings, implying that a major route of interconversion of AMP to IMP involves the reaction sequence: AMP----Ado----Ino----Hyp----IMP. PP-Rib-P generation and purine nucleoside triphosphate pools were unchanged by introduction of HGPRT deficiency into normal lymphoblast lines, in agreement with the view that accelerated purine synthesis de novo in this deficiency results from increased availability of PP-Rib-P for the pathway. Cell lines with dual enzyme defects did not differ from PP-Rib-P synthetase-superactive parental lines in rates of PP-Rib-P and purine synthesis despite 5-6-fold increases in PP-Rib-P concentrations, excretion of nearly 50% of newly synthesized purines, and diminished GTP concentrations. Fixed rates of purine synthesis de novo in PP-Rib-P synthetase-superactive cells appeared to reflect saturation of the rate-limiting amidophosphoribosyltransferase reaction for PP-Rib-P. In combination with accelerated purine excretion, increased channeling of newly formed purines into adenylates, and impaired conversion of AMP to IMP, fixed rates of purine synthesis de novo may condition cell lines with defects in HGPRT and PP-Rib-P synthetase to depletion of GTP with consequent growth retardation.  相似文献   

3.
Previous studies of purine nucleotide synthesis de novo have suggested that major regulation of the rate of the pathway is affected at either the phosphoribosylpyrophosphate (PP-Rib-P) synthetase reaction or the amidophosphoribosyltransferase (amido PRT) reaction, or both. We studied control of purine synthesis de novo in cultured normal, hypoxanthine-guanine phosphoribosyltransferase (HGPRT)-deficient, and PP-Rib-P synthetase-superactive human fibroblasts by measuring concentrations and rates of synthesis of PP-Rib-P and purine nucleotide end products, proposed effectors of regulation, during inhibition of the pathway. Incubation of cells for 90 min with 0.1 mM azaserine, a glutamine antagonist which specifically blocked the pathway at the level of conversion of formylglycinamide ribotide, resulted in a 5-16% decrease in purine nucleoside triphosphate concentrations but no consistent alteration in generation of PP-Rib-P. During this treatment, however, rates of the early steps of the pathway were increased slightly (9-15%) in normal and HGPRT-deficient strains, more markedly (32-60%) in cells with catalytically superactive PP-Rib-P synthetases, and not at all in fibroblasts with purine nucleotide feedback-resistant PP-Rib-P synthetases. In contrast, glutamine deprivation, which inhibited the pathway at the amido PRT reaction, resulted in time-dependent nucleoside triphosphate pool depletion (26-43% decrease at 24 h) accompanied by increased rates of PP-Rib-P generation and, upon readdition of glutamine, substantial increments in rates of purine synthesis de novo. Enhanced PP-Rib-P generation during glutamine deprivation was greatest in cells with regulatory defects in PP-Rib-P synthetase (2-fold), but purine synthesis in these cells was stimulated only 1.4-fold control rates by glutamine readdition. Stimulation of these processes in normal and HGPRT-deficient cells and in cells with PP-Rib-P synthetase catalytic defects was, respectively: 1.5 and 2.0-fold; 1.5 and 1.7-fold; and 1.6 and 4.1-fold. These studies support the following concepts. 1) Rates of purine synthesis de novo are regulated at both the PP-Rib-P synthetase and amido PRT reactions by end products, with the latter reaction more sensitive to small changes in purine nucleotide inhibitor concentrations. 2) PP-Rib-P exerts its role as a major regulator of purine synthetic rate by virtue of its interaction with nucleotide inhibitors to determine the activity of amido PRT. 3) Activation of amido PRT by PP-Rib-P is nearly maximal at base line in fibroblasts with regulatory defects in PP-Rib-P synthetase.  相似文献   

4.
Protective effects of exendin-4 (glucagon-like peptide-1 -GLP-1- receptor agonist) and des-fluoro-sitagliptin (dipeptidyl peptidase-4 inhibitor) on fructose-induced hepatic disturbances were evaluated in prediabetic rats. Complementary, a possible direct effect of exendin-4 in human hepatoblastoma-derived cell line HepG2 incubated with fructose in presence/absence of exendin-9-39 (GLP-1 receptor antagonist) was investigated. In vivo, after 21 days of fructose rich diet, we determined: glycemia, insulinemia, and triglyceridemia; hepatic fructokinase, AMP-deaminase, and G-6-P dehydrogenase (G-6-P DH) activities; carbohydrate-responsive element-binding protein (ChREBP) expression; triglyceride content and lipogenic gene expression (glycerol-3-phosphate acyltransferase -GPAT-, fatty acid synthase -FAS-, sterol regulatory element-binding protein-1c -SREBP-1c); oxidative stress and inflammatory markers expression. In HepG2 cells we measured fructokinase activity and triglyceride content. Hypertriglyceridemia, hyperinsulinemia, enhanced liver fructokinase, AMP-deaminase, and G-6-P DH activities, increased ChREBP and lipogenic genes expression, enhanced triglyceride level, oxidative stress and inflammatory markers recorded in fructose fed animals, were prevented by co-administration of either exendin-4 or des-fluoro-sitagliptin. Exendin-4 prevented fructose-induced increase in fructokinase activity and triglyceride contain in HepG2 cells. These effects were blunted co-incubating with exendin-9-39. The results demonstrated for the first time that exendin-4/des-fluro-sitagliptin prevented fructose-induced endocrine-metabolic oxidative stress and inflammatory changes probably acting on the purine degradation pathway. Exendin 9–39 blunted in vitro protective exendin-4 effects, thereby suggesting a direct effect of this compound on hepatocytes through GLP-1 receptor. Direct effect on fructokinase and AMP-deaminase activities, with a key role in the pathogenesis of liver dysfunction induced by fructose, suggests purine degradation pathway constitute a potential therapeutic objective for GLP-1 receptor agonists.  相似文献   

5.
Recent studies have shown that pyrroline 5-carboxylate, the intermediate in the interconversions of proline, ornithine, and glutamate, can regulate the metabolism of erythrocytes. We now report that the formation of 5-phosphoribosyl 1-pyrophosphate (PP-Rib-P) was markedly stimulated by pyrroline 5-carboxylate in intact red cells. The production of PP-Rib-P is an important point of regulation in nucleotide metabolism. We found that pyrroline 5-carboxylate increased glucose metabolism through the oxidative arm of the pentose shunt, ribose 5-phosphate formation, and PP-Rib-P production and subsequently augmented purine nucleotide production through the salvage pathway in erythrocytes. We now report that pyrroline 5-carboxylate markedly stimulated the net synthesis of inosine monophosphate from hypoxanthine in intact human red cells so that the pool of inosine monophosphate became 20-30% of the total pool of purine nucleotides. Inosine monophosphate has been considered to be a "mobile pool" of purines, i.e. a reservoir from which peripheral tissues can be supplied; the effect of pyrroline 5-carboxylate on the inosine monophosphate pool may be a mechanism for regulating the function of erythrocytes in purine delivery.  相似文献   

6.
The effect of increasing cellular ribose 5-phosphate (ribose-5-P) availability by methylene blue-induced acceleration of the oxidative pentose phosphate pathway on the rate of 5-phosphoribosyl-1-pyrophosphate (P-ribose-PP) generation, was studied in slices of rat liver at varying Pi concentration. It was found that at Pi concentration prevailing in the tissue of extracellular physiological Pi concentration, ribose-5-P availability is saturating for P-ribose-PP generation, as gauged by the rate of adenine incorporation into tissue nucleotides. The effect of altering P-ribose-PP availability on the rate of de novo purine production gauged by the rate of formate incorporation into purines, was also studied. It was found that the physiological P-ribose-PP concentration in rat liver tissue is limiting for purine synthesis de novo. Depletion of cellular P-ribose-PP, achieved by increase of P-ribose-PP consumption, decelerated purine synthesis, while increase of P-ribose-PP availability, achieved by activation of P-ribose-PP synthetase occurring at elevated Pi concentration, resulted in acceleration of purine synthesis.  相似文献   

7.
Glycogen synthesis in isolated hepatocytes can occur from glucose both by a direct mechanism and by an indirect process in which glucose is first metabolized to C3 intermediates before use for glycogenesis via gluconeogenesis. We studied the incorporation into glycogen of glucose and the gluconeogenic substrate, fructose, in primary cultures of hepatocytes from fasted rats. In the presence of insulin, both glucose and fructose promoted net deposition of glycogen; however, fructose carbon was incorporated into glycogen to a greater extent than that from glucose. When glucose and fructose were administered simultaneously, the glycogenic utilization of glucose was stimulated 2-3-fold, and that of fructose was increased by about 50%. At constant hexose concentrations, the total incorporation of carbon, and the total accumulation of glycogen mass, from glucose and fructose when present together exceeded that from either substrate alone. Fructose did not change the relative proportion of glucose carbon incorporated into glycogen via the indirect (gluconeogenic) mechanism. The synergism of glucose and fructose in glycogen synthesis in isolated rat hepatocytes in primary culture appears to result from a decrease in the rate of degradation of newly deposited glycogen, owing to (i) decreased amount of phosphorylase a mediated by glucose and (ii) noncovalent inhibition of residual phosphorylase activity by some intermediate arising from the metabolism of fructose, presumably fructose 1-phosphate.  相似文献   

8.
Fructose-induced adenine nucleotide catabolism in isolated rat hepatocytes   总被引:1,自引:0,他引:1  
The mechanism of fructose-induced nucleotide catabolism was studied using isolated rat hepatocytes in which the adenine nucleotide pool was prelabelled with [14C]adenine. Incubation of these cells with fructose caused a rapid depletion of the adenine nucleotides and a corresponding increase in allantoin. There was no accumulation of radioactivity in adenosine in the presence or absence of the adenosine deaminase inhibitor 9-erythro-(2-hydroxy-3-nonyl)adenine. This confirms the previous hypothesis that fructose-induced adenine nucleotide catabolism occurs by way of AMP deaminase (AMP amino-hydrolase, EC 3.5.4.6).  相似文献   

9.
Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease in the world, and it is thought to be the hepatic manifestation of the metabolic syndrome. Excess dietary fructose causes both metabolic syndrome and NAFLD in rodents and humans, but the pathogenic mechanisms of fructose-induced metabolic syndrome and NAFLD are poorly understood. GLUT8 (Slc2A8) is a facilitative glucose and fructose transporter that is highly expressed in liver, heart, and other oxidative tissues. We previously demonstrated that female mice lacking GLUT8 exhibit impaired first-pass hepatic fructose metabolism, suggesting that fructose transport into the hepatocyte, the primary site of fructose metabolism, is in part mediated by GLUT8. Here, we tested the hypothesis that GLUT8 is required for hepatocyte fructose uptake and for the development of fructose-induced NAFLD. We demonstrate that GLUT8 is a cell surface-localized transporter and that GLUT8 overexpression or GLUT8 shRNA-mediated gene silencing significantly induces and blocks radiolabeled fructose uptake in cultured hepatocytes. We further show diminished fructose uptake and de novo lipogenesis in fructose-challenged GLUT8-deficient hepatocytes. Finally, livers from long term high-fructose diet-fed GLUT8-deficient mice exhibited attenuated fructose-induced hepatic triglyceride and cholesterol accumulation without changes in hepatocyte insulin-stimulated Akt phosphorylation. GLUT8 is thus essential for hepatocyte fructose transport and fructose-induced macrosteatosis. Fructose delivery across the hepatocyte membrane is thus a proximal, modifiable disease mechanism that may be exploited to prevent NAFLD.  相似文献   

10.
Incubation of freshly isolated rat liver cells with glycerol resulted in an initial decrease, followed by an increase in purine synthesis de novo and in PP-ribose-P availability. The magnitude of these effects was dependent on the concentration of glycerol; as it increased, the initial period of latency or inhibition was prolonged, and the extent of the subsequent stimulation was greater. The intracellular Pi concentration and the [14C]ATP/[14C]ADP ratio were also initially decreased in these cells, and they too returned subsequently to normal values. All these changes were similar to those induced by fructose under the same conditions. The increase in PP-ribose-P availability always preceded that in purine synthesis de novo, indicating that, under most circumstances, PP-ribose-P availability is limiting for purine synthesis de novo. Finally, PP-ribose-P synthesis in these cells varied in parallel with the intracellular Pi concentration and with the ATP/ADP and ATP/AMP ratios.  相似文献   

11.
12.
De novo synthesis of purine nucleotides and some regulatory properties of this pathway were studied in cultured epithelial-like rat liver cells. It was found that the physiological 5-phosphoribosyl 1-pyrophosphate (P-Rib-PP) concentration in these cells is limiting for purine synthesis de novo. Increase of P-Rib-PP availability, achieved by activation of P-Rib-PP synthetase at high Pi concentration, resulted in acceleration of purine synthesis. The effects of increasing cellular ribose 5-phosphate (Rib-5-P availability, by methylene blue-induced acceleration of the oxidative pentose phosphate pathway, on P-Rib-PP availability and on the rate of the novo purine synthesis were also studied. It was found that at the Pi concentration prevailing in the tissue at extracellular physiological Pi concentration, Rib-5-P availability is saturating for P-Rib-PP generation and therefore also for purine synthesis.  相似文献   

13.
Deoxyadenosine (dAdo) and deoxyguanosine (dGuo) decrease methionine synthesis from homocysteine in cultured lymphoblasts; because of the possible trapping of 5-methyltetrahydrofolate this could lead to decreased purine nucleotide synthesis. Since purine deoxynucleosides could also inhibit purine synthesis de novo at an early step not involving folate metabolism, we measured in azaserine-treated cells 5-amino-4-imidazolecarboxamide (Z-base)-dependent purine nucleotide synthesis using [14C]formate. In the T lymphoblasts, Z-base-dependent purine nucleotide synthesis was decreased 26% by 0.3 microM-dAdo, 21% by 1 microM-dGuo and 28% by 1 microM-adenosine dialdehyde, a potent S-adenosylhomocysteine hydrolase inhibitor; homocysteine fully reversed the inhibitions. The B lymphoblasts were considerably less sensitive to the deoxynucleoside-induced decrease in Z-base-dependent purine nucleotide synthesis, with 100 microM-dAdo required for significant inhibition and no inhibition by dGuo at this concentration; homocysteine partly reversed the inhibition by dAdo. The observed decrease in Z-base-dependent purine nucleotide synthesis could not be attributed either to dUMP depletion changing the folate pools or to decreased ATP availability because dUrd was without effect and during the experimental period the intracellular ATP concentration did not change significantly. Cells with 5,10-methylenetetrahydrofolate reductase deficiency were relatively resistant to inhibition of Z-base-dependent purine nucleotide synthesis by dAdo and adenosine dialdehyde. Our results suggest that deoxynucleosides decrease purine nucleotide synthesis by trapping 5-methyltetrahydrofolate.  相似文献   

14.
Lesch-Nyhan syndrome is a pediatric metabolic-neurological syndrome caused by the X-linked deficiency of the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT). The cause of the metabolic consequences of HGPRT deficiency has been clarified, but the connection between the enzyme deficiency and the neurological manifestations is still unknown. In search for this connection, in the present study, we characterized purine nucleotide metabolism in primary astroglia cultures from HGPRT-deficient transgenic mice. The HGPRT-deficient astroglia exhibited the basic abnormalities in purine metabolism reported before in neurons and various other HGPRT-deficient cells. The following abnormalities were found: absence of detectable uptake of guanine and of hypoxanthine into intact cell nucleotides; 27.8% increase in the availability of 5-phosphoribosyl-1-pyrophosphate; 9.4-fold acceleration of the rate of de novo nucleotide synthesis; manyfold increase in the excretion into the culture media of hypoxanthine (but normal excretion of xanthine); enhanced loss of label from prelabeled adenine nucleotides (loss of 71% in 24 h, in comparison with 52.7% in the normal cells), due to 4.2-fold greater excretion into the media of labeled hypoxanthine. In addition, the HGPRT-deficient astroglia were shown to contain lower cellular levels of ADP, ATP, and GTP, indicating that the accelerated de novo purine synthesis does not compensate adequately for the deficiency of salvage nucleotide synthesis, and higher level of UTP, probably due to enhanced de novo synthesis of pyrimidine nucleotides. Altered nucleotide content in the brain may have a role in the pathogenesis of the neurological deficit in Lesch-Nyhan syndrome.  相似文献   

15.
Mouse hepatocytes from healthy control mice and from Ehrlich ascites tumour-bearing mice were used for tracer-kinetic studies of purine catabolism of liver cells during different periods of tumour growth. The dynamics of the radioactive tracers were modelled mathematically by a system of differential equations. Computer simulations, i.e. direct fitting of numerical solutions of these equations to the observed time-courses of metabolites and specific radioactivites, enables one to estimate unknown kinetic parameters of a simplified model of pathways of hepatic purine catabolism in tumour-bearing mice. There occurred great differences of metabolic flux rates between control hepatocytes, hepatocytes of mice during the proliferating period of tumour growth (6th day after inoculation of the tumour) and hepatocytes of mice during the resting period of tumour growth (12th day after inoculation of the tumour). The final purine degradation of hepatocytes prepared during the proliferating period was lower in comparison with that of control hepatocytes, but it was markedly higher in hepatocytes prepared during the resting period of tumour growth. The changes in hepatocyte purine catabolism during the proliferating period of tumour growth argue for transitions which aim at the maintenance of high purine nucleotide levels in the liver itself rather than for an increased nucleoside and nucleobase supply for the tumour. This suggestion is in accordance with the increased ATP level of the liver during the proliferating phase of tumour growth. The drastic acceleration of the final steps of hepatic purine catabolism forming uric acid and allantoin during the resting period of tumour growth was predominantly due to increased flux rate from xanthosine and guanine in accordance with increased catabolism of monophosphorylated nucleotides.  相似文献   

16.
The mechanism for glycogen synthesis stimulation produced by adenosine, fructose, and glutamine has been investigated. We have analyzed the relationship between adenine nucleotides and glycogen metabolism rate-limiting enzymes upon hepatocyte incubation with these three compounds. In isolated hepatocytes, inhibition of AMP deaminase with erythro-9-(2-hydroxyl-3nonyl)adenine further increases the accumulation of AMP and the activation of glycogen synthase and phosphorylase by fructose. This ketose does not increase cyclic AMP or the activity of cyclic AMP-dependent protein kinase. Adenosine raises AMP and ATP concentration. This nucleotide also activates glycogen synthase and phosphorylase by covalent modification. The correlation coefficient between AMP and glycogen synthase activity is 0.974. Nitrobenzylthioinosine, a transport inhibitor of adenosine, blocks (by 50%) the effect of the nucleoside on AMP formation and glycogen synthase but not on phosphorylase. 2-Chloroadenosine and N6-phenylisopropyladenosine, nonmetabolizable analogues of adenosine, activate phosphorylase (6-fold) without increasing the concentration of adenine nucleotides or the activity of glycogen synthase. Cyclic AMP is not increased by adenosine in hepatocytes from starved rats but is in cells from fed animals. [Ethylenebis (oxyethylenenitrilo)]tetraacetic acid (EGTA) blocks by 60% the activation of phosphorylase by adenosine but not that of glycogen synthase. Glutamine also increases AMP concentration and glycogen synthase and phosphorylase activities, and these effects are blocked by 6-mercaptopurine, a purine synthesis inhibitor. Neither adenosine nor glutamine increases glucose 6-phosphate. It is proposed that the observed efficient glycogen synthesis from fructose, adenosine, and glutamine is due to the generation of AMP that activates glycogen synthase probably through increases in synthase phosphatase activity. It is also concluded that the activation of phosphorylase by the above-mentioned compounds can be triggered by metabolic changes.  相似文献   

17.
The ability of Zn to modulate key metabolic processes was investigated in a study of gluconeogenesis in isolated hepatocytes from fasted rats. Zn (100 μM) inhibited glucose production from fructose by 41%, sorbitol by 28%; glycerol by 17%, and glyceraldehyde by 26%. Maximum inhibition of gluconeogenesis from fructose occurred at 25 μM Zn. Zn inhibited the rate of lactate production from fructose by 24% but not from sorbitol, glycerol, or glyceraldehyde. Fructose uptake by hepatocytes was not affected by Zn. A positive linear relationship (r=0.994) was obtained between inhibition by Zn of glucose and lactate production, indicating that a common step in both pathways is inhibited by Zn. The effect of Zn on fructokinase, aldolase-B, and triokinase activities was determined on semipurified rat liver enzyme preparations. Zn had no affect on triokinase activity but inhibited the two other enzymes in a dose-dependent manner, with the inhibition of aldolase-B being much greater than of fructokinase for concentrations of Zn between 2.5 and 20 μM. Zn increased the intracellular concentration of fructose-1-P in hepatocytes incubated with fructose, indicating a more potent Zn inhibition of aldolase-B than fructokinase. In addition, hepatocytes treated with Zn had decreased ATP and ADP concentrations, but had normal energy charge, suggesting an effect of Zn on adenine nucleotide degradation or synthesis. The demonstration that Zn inhibits two enzymes in fructose metabolism adds to the growing list of metabolic pathways that are catalyzed by enzymes that are sensitive to Zn.  相似文献   

18.
19.
20.
Pathways of purine nucleotide metabolism affecting the availability of ATP in the muscle tissue were studied in differentiating rat muscle cultures. The rate of de novo purine nucleotide synthesis and of AMP deamination were found to increase markedly with cell differentiation, but the rate of IMP dephosphorylation was similarly low in both myoblasts and contracting fibers. The above differentiation-associated alterations in purine nucleotide metabolism conform with the greater need for ATP as a source of energy in the contracting myotubes.  相似文献   

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