Elsevier

Biochemical Pharmacology

Volume 52, Issue 7, 11 October 1996, Pages 999-1006
Biochemical Pharmacology

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

https://doi.org/10.1016/0006-2952(96)00413-3Get rights and content

Abstract

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 μM 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 μmol/g of cells). Dephosphorylation of ZMP added to crude cytosolic extracts of rat liver displayed hyperbolic kinetics, with a Vmax of 0.65 μmol/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.

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      Indeed, AMPK is activated by conditions that increase intracellular AMP such as hypoglycemia, hypoxia and exercise, as well as certain cytokines and drugs, such as metformin and 5-aminoimidazole-4-carboxamide riboside (AICA riboside or AICAR) [21]. AICAR is a cell-permeable nucleoside which could be metabolically converted to AICA ribotide (or ZMP), the antepenultimate metabolic intermediate of the de novo purine synthesis pathway [22]. The liver kinase B1 (LKB1) and Ca2+/calmodulin-dependent protein kinase kinase-β (CaMKKβ) are upstream kinases that activate AMPK by phosphorylating Thr172 in the activation loop of the catalytic α-subunits [23,24].

    • Phenotypes and circadian rhythm in utilization of formate in purine nucleotide biosynthesis de novo in adult humans

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      The above evidence indicates that PNB in mammalian liver stops at AICAR or at an earlier step. Since substrate cycling of AICAR to AICA-riboside (which crosses cell membranes) occurs in isolated rat hepatocytes (Vincent et al., 1996), and AICA, an AICAR metabolite, is normally found in human urine (McGreer et al., 1961), PNB in human liver probably stops at AICAR; therefore, its riboside is in circulation. AICAR to IMP metabolism should occur in rat liver since the specific activity of AICAR transformylase is greater than that of GAR transformylase (Deacon et al., 1985).

    • <sup>13</sup>C enrichment of carbons 2 and 8 of purine by folate-dependent reactions after [<sup>13</sup>C]formate and [2-<sup>13</sup>C]glycine dosing in adult humans

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      Our finding differs from the previously reported C2/C8 ratio of about 1.0 in animals [6-8]. We postulate that the liver stops purine biosynthesis de novo at AICAR because isolated mammalian hepatocytes do not metabolize AICAR to inosine monophosphate (IMP) or to any other purines [24-28]. This is consistent with the low capacity of the mammalian hepatocytes or liver slices to synthesize purines in vitro from radioactive formate or serine [29-31].

    • AMP-activated protein kinase and the regulation of autophagic proteolysis

      2006, Journal of Biological Chemistry
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      The ability of compound C, in contrast to amino acids, to inhibit AICAR-induced AMPK phosphorylation in hepatocytes on its own (Fig. 4), even though the accumulation of ZMP in both cases decreased similarly (Table 1) may be ascribed to small differences in intra/extracellular distribution of ZMP. Although most of the ZMP produced from AICAR is recovered inside the hepatocytes (50), it is possible that in the presence of amino acids, when cell volume increases (8), some of the ZMP leaks to the extracellular fluid so that the total amount of ZMP in the cell suspension overestimates the actual concentration of ZMP inside the cells. In summary, the data presented in this study confirm that the AMPK activator AICAR inhibits autophagy but do not support the conclusion that AMPK is responsible for this effect (11).

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    We thank T. Timmerman for expert technical assistance. This work was supported by an endowment from Gensia Inc., San Diego, CA, USA, by grant 3.4557.93 of the Belgian Fund for Medical Scientific Research, and by the Belgian Federal Service for Scientific, Technical and Cultural Affairs. G.V.D.B. is Research Director of the Belgian National Fund for Scientific Research.

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