scholarly journals AMP-activated protein kinase-independent inhibition of hepatic mitochondrial oxidative phosphorylation by AICA riboside

2007 ◽  
Vol 404 (3) ◽  
pp. 499-507 ◽  
Author(s):  
Bruno Guigas ◽  
Nellie Taleux ◽  
Marc Foretz ◽  
Dominique Detaille ◽  
Fabrizio Andreelli ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Oxidative Phosphorylation ◽  
Adenine Nucleotide ◽  
Adenine Nucleotides ◽  
Cellular Respiration ◽  
Mitochondrial Oxidative Phosphorylation ◽  
Isolated Rat Hepatocytes ◽  
Amp Activated Protein Kinase ◽  
Isolated Rat ◽  
In Cells

AICA riboside (5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside) has been extensively used in cells to activate the AMPK (AMP-activated protein kinase), a metabolic sensor involved in cell energy homoeostasis. In the present study, we investigated the effects of AICA riboside on mitochondrial oxidative; phosphorylation. AICA riboside was found to dose-dependently inhibit the oligomycin-sensitive JO2 (oxygen consumption rate) of isolated rat hepatocytes. A decrease in Pi (inorganic phosphate), ATP, AMP and total adenine nucleotide contents was also observed with AICA riboside concentrations >0.1 mM. Interestingly, in hepatocytes from mice lacking both α1 and α2 AMPK catalytic subunits, basal JO2 and expression of several mitochondrial proteins were significantly reduced compared with wild-type mice, suggesting that mitochondrial biogenesis was perturbed. However, inhibition of JO2 by AICA riboside was still present in the mutant mice and thus was clearly not mediated by AMPK. In permeabilized hepatocytes, this inhibition was no longer evident, suggesting that it could be due to intracellular accumulation of Z nucleotides and/or loss of adenine nucleotides and Pi. ZMP did indeed inhibit respiration in isolated rat mitochondria through a direct effect on the respiratory-chain complex I. In addition, inhibition of JO2 by AICA riboside was also potentiated in cells incubated with fructose to deplete adenine nucleotides and Pi. We conclude that AICA riboside inhibits cellular respiration by an AMPK-independent mechanism that likely results from the combined intracellular Pi depletion and ZMP accumulation. Our data also demonstrate that the cellular effects of AICA riboside are not necessarily caused by AMPK activation and that their interpretation should be taken with caution.

scholarly journals Purine catabolism in isolated rat hepatocytes. Influence of coformycin

1980 ◽  
Vol 188 (3) ◽  
pp. 913-920 ◽  
Author(s):  
Georges Van Den Berghe ◽  
Françoise Bontemps ◽  
Henri-Géry Hers
Keyword(s):  
High Speed ◽  
Adenine Nucleotide ◽  
Adenine Nucleotides ◽  
Rat Hepatocytes ◽  
Amp Deaminase ◽  
Purine Nucleotides ◽  
Isolated Rat Hepatocytes ◽  
Adenine Nucleotide Pool ◽  
Isolated Rat

1. The catabolism of purine nucleotides was investigated by both chemical and radiochemical methods in isolated rat hepatocytes, previously incubated with [14C]adenine. The production of allantoin reached 32±5nmol/min per g of cells (mean±s.e.m.) and as much as 30% of the radioactivity incorporated in the adenine nucleotides was lost after 1h. This rate of degradation is severalfold in excess over values previously reported to occur in the liver in vivo. An explanation for this enhancement of catabolism may be the decrease in the concentration of GTP. 2. In a high-speed supernatant of rat liver, adenosine deaminase was maximally inhibited by 0.1μm-coformycin. The activity of AMP deaminase, measured in the presence of its stimulator ATP in the same preparation, as well as the activity of the partially purified enzyme, measured after addition of its physiological inhibitors GTP and Pi, required 50μm-coformycin for maximal inhibition. 3. The production of allantoin by isolated hepatocytes was not influenced by the addition of 0.1μm-coformycin, but was decreased by concentrations of coformycin that were inhibitory for AMP deaminase. With 50μm-coformycin the production of allantoin was decreased by 85% and the formation of radioactive allantoin from [14C]adenine nucleotides was completely suppressed. 4. In the presence of 0.1μm-coformycin or in its absence, the addition of fructose (1mg/ml) to the incubation medium caused a rapid degradation of ATP, without equivalent increase in ADP and AMP, followed by transient increases in IMP and in the rate of production of allantoin; adenosine was not detectable. In the presence of 50μm-coformycin, the fructose-induced breakdown of ATP was not modified, but the depletion of the adenine nucleotide pool proceeded much more slowly and the rate of production of allantoin increased only slightly. No rise in IMP concentration could be detected, but AMP increased manyfold and reached values at which a participation of soluble 5′-nucleotidase in the catabolism of adenine nucleotides is most likely. 5. These results are in agreement with the hypothesis that the formation of allantoin is controlled by AMP deaminase. They constitute further evidence that 5′-nucleotidase is inactive on AMP, unless the concentration of this nucleotide rises to unphysiological values.

scholarly journals Okadaic acid-induced, naringin-sensitive phosphorylation of glycine N-methyltransferase in isolated rat hepatocytes

2003 ◽  
Vol 373 (2) ◽  
pp. 505-513 ◽  
Author(s):  
Michael T. N. MØLLER ◽  
Hamid R. SAMARI ◽  
Monica FENGSRUD ◽  
Per E. STRØMHAUG ◽  
Anne C. ØSTVOLD ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Okadaic Acid ◽  
Rat Hepatocytes ◽  
Isolated Rat Hepatocytes ◽  
Limited Region ◽  
Flight Mass Spectrometry ◽  
Methylation Capacity ◽  
Dependent Protein ◽  
Amp Activated Protein Kinase ◽  
Isolated Rat

Glycine N-methyltransferase (GNMT) is an abundant cytosolic enzyme that catalyses the methylation of glycine into sarcosine, coupled with conversion of the methyl donor, S-adenosylmethionine (AdoMet), into S-adenosylhomocysteine (AdoHcy). GNMT is believed to play a role in monitoring the AdoMet/AdoHcy ratio, and hence the cellular methylation capacity, but regulation of the enzyme itself is not well understood. In the present study, treatment of isolated rat hepatocytes with the protein phosphatase inhibitor okadaic acid, was found to induce an overphosphorylation of GNMT, as shown by proteomic analysis. The analysis comprised two-dimensional gel electrophoretic separation of 32P-labelled phosphoproteins and identification of individual protein spots by matrix-assisted laser-desorption ionization–time-of-flight mass spectrometry. The identity of GNMT was verified by N-terminal Edman sequencing of tryptic peptides. Chromatographic separation of proteolytic peptides and 32P-labelled amino acids suggested that GNMT was phosphorylated within a limited region, and only at serine residues. GNMT phosphorylation could be suppressed by naringin, an okadaic acid-antagonistic flavonoid. To assess the possible functional role of GNMT phosphorylation, the effect of okadaic acid on hepatocytic AdoMet and AdoHcy levels was examined, using HPLC separation for metabolite analysis. Surprisingly, okadaic acid was found to have no effect on the basal levels of AdoMet or AdoHcy. An accelerated AdoMet–AdoHcy flux, induced by the addition of methionine (1 mM), was likewise unaffected by okadaic acid. 5-Aminoimidazole-4-carboxamide riboside, an activator of the hepatocytic AMP-activated protein kinase, similarly induced GNMT phosphorylation without affecting AdoMet and AdoHcy levels. Activation of cAMP-dependent protein kinase by dibutyryl-cAMP, reported to cause GNMT phosphorylation under cell-free conditions, also had little effect on hepatocytic AdoMet and AdoHcy levels. Phosphorylation of GNMT would thus seem to play no role in regulation of the intracellular AdoMet/AdoHcy ratio, but could be involved in other GNMT functions, such as the binding of folates or aromatic hydrocarbons.

Fructose-induced adenine nucleotide catabolism in isolated rat hepatocytes

1977 ◽  
Vol 55 (12) ◽  
pp. 1237-1240 ◽  
Author(s):  
Camilla M. Smith ◽  
Liisa M. Rovamo ◽  
Kari O. Raivio
Keyword(s):  
Adenosine Deaminase ◽  
Adenine Nucleotide ◽  
Adenine Nucleotides ◽  
Rat Hepatocytes ◽  
Amp Deaminase ◽  
Isolated Rat Hepatocytes ◽  
Previous Hypothesis ◽  
Adenine Nucleotide Pool ◽  
Isolated Rat

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).

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