Cytosolic 5′-Nucleotidase II Silencing in Lung Tumor Cells Regulates Metabolism through Activation of the p53/AMPK Signaling Pathway

Cytosolic 5′-nucleotidase II (cN-II) is an allosteric catabolic enzyme that hydrolyzes IMP, GMP, and AMP. The enzyme can assume at least two different structures, being the more active conformation stabilized by ATP and the less active by inorganic phosphate. Therefore, the variation in ATP concentration can control both structure and activity of cN-II. In this paper, using a capillary electrophoresis technique, we demonstrated that a partial silencing of cN-II in a pulmonary carcinoma cell line (NCI-H292) is accompanied by a decrease in adenylate pool, without affecting the energy charge. We also found that cN-II silencing decreased proliferation and increased oxidative metabolism, as indicated by the decreased production of lactate. These effects, as demonstrated by Western blotting, appear to be mediated by both p53 and AMP-activated protein kinase, as most of them are prevented by pifithrin-α, a known p53 inhibitor. These results are in line with our previous observations of a shift towards a more oxidative and less proliferative phenotype of tumoral cells with a low expression of cN-II, thus supporting the search for specific inhibitors of this enzyme as a therapeutic tool for the treatment of tumors.

Exploring the Biochemical Basis of the Meridian Tropism Theory for the Qi-Invigorating Traditional Chinese Medicine Herb Panax ginseng

We examined the effect of the Qi-invigorating Traditional Chinese Medicines (TCM) herb Panax ginseng ( P.G.) on mitochondrial functions and cellular antioxidant capacity in different organs of mice. We found that the P.G. extracts had a significant effect on tissues of mice, with the generation of total adenylate pool (TAP) enhanced in all visceral tissues, but not for the brain. The mitochondrial membrane potential (MMP) and antioxidant capacity reflected by superoxide dismutase (SOD) and glutathione (GSH) increased only for the meridian tissues that P.G. belongs to including Heart, Spleen and Lung. Reactive oxygen species (ROS), as a combined result of the increased energy metabolism and antioxidant capacity, varied in different organs. We concluded that: 1) the Qi-invigorating TCM herb P.G. had a significant effect on mice by enhancing TAP production in all of the visceral tissues examined, except for the brain; 2) for the meridional tissues of P.G. (Heart, Spleen and Lung), the P.G. extracts not only promoted the TAP production, but also boosted the antioxidant capacity demonstrated by the simultaneous increase in TAP, and SOD and GSH.

Modulation of adenine phosphoribosyltransferase-mediated salvage to promote diabetic wound healing

Adenine phosphoribosyltransferase (APRT) is the key enzyme in purine salvage by the incorporation of adenine and phosphoribosyl pyrophosphate to provide adenylate nucleotide. The up-regulated APRT found in wound skin correlated with the demands of repair in diabetic mice. Administration of adenine on the wound of diabetic mice exhibited elevated ATP levels in organismic skin and accelerated wound healing. In vitro studies showed that APRT utilized adenine to rescue cellular ATP levels and proliferation against hydrogen peroxide-induced oxidative damage. LC-MS/MS-based analysis of total adenylate nucleotides in NIH-3T3 fibroblast showed that adenine addition enlarged the cellular adenylate pool, reduced the adenylate energy charge, and provided more AMP for the generation of ATP in further. These data indicated the role of APRT during diabetic wound healing by regulating the nucleotide pool after injury and demonstrated the improvement by topical adenine, which highlights its value as a promising agent in therapeutic intervention. Our study provided an explanation for the up- regulation of APRT in tissue repair and adenine supplement resulted in an enlargement of the adenylate pool for ATP generation.

Extracellular Degradation into Adenosine and the Activities of Adenosine Kinase and AMPK Mediate Extracellular NAD+-produced increases in the Adenylate Pool of BV2 Microglia under Basal Conditions

Cumulating evidence has indicated NAD+ deficiency as a common central pathological factor of multiple diseases and aging. NAD+ supplement is highly protective in various disease and aging models, while two key questions remain unanswered: 1) Does extracellular NAD+ also produce its effects through its degradation product adenosine? 2) Does extracellular NAD+ produce the protective effects by affecting cells under pathological insults only, or by affecting both normal cell and cells under pathological insults? Since extracellular NAD+ can be degraded into adenosine, and endogenous adenosine levels are in the nanomolar range under physiological conditions, extracellular NAD+ may produce its effects through its degradation into adenosine. In this study we used BV2 microglia as a cellular model to test our hypothesis that NAD+ treatment can increase the intracellular adenylate pool under basal conditions through its extracellular degradation into adenosine. Our study has shown that extracellular NAD+ increases the adenylate pool of BV2 microglia under basal conditions through its degradation into adenosine that enters the cells through equilibrative nucleoside transporters. The intracellular adenosine is converted to AMP by adenosine kinase, which increases intracellular ATP by both activating AMPK and increasing ADP that drives mitochondrial FoF1-ATP synthase. Collectively, our study has suggested that extracellular NAD+ can enhance defensive capacity of normal cells through a novel pathway, which includes extracellular NAD+ degradation into adenosine and the activities of adenosine kinase and AMPK. Our findings have also suggested that NAD+ administration in various disease and aging models may significantly affect the microglia under basal conditions.

What Determines the Intracellular ATP Concentration

Analysis is made of the mechanisms that control the intracellular ATP level. The balance between energy production and expenditure determines the energy charge of the cell and the ratio of [ATP] to the adenylate pool. The absolute ATP concentration is determined by the adenylate pool, which, in its turn, depends on the balance between the rates of AMP synthesis and degradation. Experimental data are discussed that demonstrate an increase in the adenylate pool in response to activation of energy-consuming processes. A hypothesis is proposed according to which variation in the adenylate pool and absolute ATP concentration affords a cell the possibility of additional control over processes fulfilling useful work. A mechanism involved in this regulation is described using human erythrocytes as an example. The hypothesis explains why different metabolic pathways (protein and DNA syntheses, polysaccharide synthesis, and lipid synthesis) use different trinucleotides (GTP, UTP, and CTP, respectively) as an energy source. This allows the cell to independently control these metabolic processes by varying the individual nucleotide pools.

Elevation of the adenylate pool in rat cardiomyocytes by S-adenosyl-L-methionine.

Rapid resynthesis of the adenylate pool in cardiac myocytes is important for recovery of contractility and normal function of regulatory mechanisms in the heart. Adenosine and adenine are thought to be the most effective substrates for nucleotide synthesis, but the possibility of using other compounds has been studied very little in cardiomyocytes. In the present study, the effect of S-adenosyl-L-methionine (SAM) on the adenylate pool of isolated cardiomyocytes was investigated and compared to the effect of adenine and adenosine. Adult rat cardiomyocytes were isolated using the collagenase perfusion technique. The cells were incubated in the presence of adenine derivatives for 90 min followed by nucleotide determination by HPLC. The concentrations of adenine nucleotides expressed in nmol/mg of cell protein were initially 22.1 +/- 1.4, 4.0 +/- 0.3 and 0.70 +/- 0.08 for ATP, ADP and AMP, respectively (n = 10, +/- S.E.M.), and the total adenylate pool was 26.8 +/- 1.6. In the presence of 1.25 mM SAM in the medium, the adenylate pool increased by 5.2 +/- 0.4 nmol/mg of cell protein, but only if 1 mM ribose was additionally present in the medium. No changes were observed with SAM alone. A similar increase (by 4.9 +/- 0.6 nmol/mg protein) was observed after incubation with 1.25 mM adenine plus 1 mM ribose, but no increase was observed if ribose was omitted. Adenosine at 0.1 or 1.25 mM concentrations also caused an increase in the adenylate pool (by 5.2 +/- 1.0 and 5.2 +/- 0.9 nmol/mg protein, respectively), which in contrast to the SAM or adenine was independent of the additional presence of ribose. Thus, S-adenosyl-L-methionine could be used as a precursor of the adenylate pool in cardiomyocytes, which is as efficient in increasing the adenylate pool after 90 min of incubation as adenosine or adenine. Nucleotide synthesis from SAM involves the formation of adenine as an intermediate with its subsequent incorporation by adenine phosphoribosyltransferase.

Flight-muscle adenylate pool responses to flight demands and thermal constraints in individual Colias eurytheme (Lepidoptera, pieridae)

We study here the connections among body temperature variation, flight performance and flight ‘fuel’ metabolism in Colias eurytheme butterflies, to begin re-examining the metabolic reasons for animal thermoregulation. Methods are presented for (a) stable extraction of adenylates (and other metabolites) from the flight muscles of individual Colias eurytheme, (b) automated separation and quantitative analysis of individual adenylate samples by high-pressure liquid chromatography and (c) reliable, low-variance assay of inorganic phosphate levels in the same extracts. Correlations among metabolite concentrations and two indices of muscle cytosol ATP maintenance occur as expected on general metabolic principles. [ATP] and [ATP]/[ADP] decline from resting levels to reach a plateau in the first minute of free, interrupted flight, while [AMP] increases at the same time; these concentrations do not vary further for up to 6 min total flight time. In an initial test of the alternative metabolic bases of the thermoregulation of Colias eurytheme, we find that [ATP]/[ADP] rises between a body temperature, T(b), of 31 and 35 degrees C, at the base of the behavioral thermal optimum for flight, but then decreases again at T(b)=39 degrees C, at the top of the behavioral thermal optimum and well short of damaging temperatures. This is not consistent with the view that metabolic effectiveness increases monotonically up to the lower limits of thermal damage to enzymes, but supports an alternative hypothesis that the narrowness of thermoregulation results from a system-based constraint on the breadth of temperature over which maximal energy processing is possible.