Exogenous Mg-ATP Induces a Large Inhibition of Pyruvate Kinase in Intact Rat Hepatocytes

Abstract

Mg-ATP infusion in vivo has been reported to be beneficial both to organ function and survival rate in various models of shock. Moreover, a large variety of metabolic effects has been shown to occur in several tissues due to purinergic receptor activation. In the present work we studied the effects of exogenous Mg-ATP in rat liver cells perifused with dihydroxyacetone to investigate simultaneously gluconeogenetic and glycolytic pathways. We found a significant effect on oxidative phosphorylation as characterized by a decrease in oxygen consumption rate and in the cellular ATP-to-ADP ratio associated with an increase in lactate-to-pyruvate ratio. In addition, exogenous Mg-ATP induced rapid and reversible inhibition of both gluconeogenesis and glycolysis. The main effect on gluconeogenesis was located at the level of the fructose cycle, whereas the decrease in glycolysis was due to a strong inhibition of pyruvate kinase. Although pyruvate kinase inhibition induced by exogenous Mg-ATP was allosteric when assessed in vitro after enzyme extraction, we found a large decrease in the apparent maximal velocity when kinetics were assessed in vivo in intact perifused hepatocytes. This newly described short-term regulation of pyruvate kinase occurs only in the intact cell and may open new potentials for the pharmacological regulation of pyruvate kinase in vivo.Numerous publications have emphasized the beneficial role of Mg-ATP infusion in various models of shock or severe trauma in animals. Dysfunction of heart (1), kidney (2), muscle (3), endothelial (4), and immune cells (5, 6) are all improved by this treatment, which significantly increases the survival rate. Because such a benefit was independent of hemodynamic status (4, 7), a metabolic mechanism was proposed (8). Because the liver plays a central role in the metabolic response to such severe illnesses, it may represent a major target for Mg-ATP (9, 10).It is well known that purinergic receptor activation is responsible for a large variety of metabolic effects (11-24). ATP or adenosine or several analogues of purinergic receptors have been noted to affect liver glucose metabolism: i.e. stimulation of glycogenolysis (12, 13), increase (19,25,26) or decrease (11) of gluconeogenesis, and decrease of glycolysis (25). These effects have been related to a cAMP-dependent mechanism (12), a cAMP-independent inositol 3-phosphate/calcium-mediated signaling (13,14,16,18,21,23), a phospholipase C activation (18), or a transcriptional effect (25). It must be noted, however, that if some effects are shared among the adenine nucleotide family, others appear to be specific (12,14,18), suggesting that different signaling pathways may be involved.Because the beneficial effects were observed in vivo with Mg-ATP but not with adenosine (8, 27), we investigated the metabolic effects of exogenous Mg-ATP in rat liver cells perifused with DHA.1 Besides a significant effect on oxidative phosphorylation and on gluconeogenesis, we found that Mg-ATP was responsible for a large inhibition of pyruva...

Interrelation between the inhibition of glycolytic flux by silibinin and the lowering of mitochondrial ROS production in perifused rat hepatocytes

“…This obviously means that the enzyme content was not modified by such a short-term effect of silibinin, which is rather expected. Similar results were found with exogenous Mg-ATP infusion but, in this case, while PK inhibition was allosteric when assessed on cell extracts, the kinetics for this enzyme were profoundly affected in intact perifused hepatocytes, the relationship being no longer of allosteric type but hyperbolic, the apparent maximum being decreased by half (Ichai et al, 2001a). Adenosine was also shown to inhibit PK activity, due to a cAMP-related phosphorylation (Bartrons et al, 1984).…”

“…PK activity was assessed on resuspended cell pellets as previously reported (Ichai et al, 2001a). Briefly, the enzyme was first incubated at 22°C for 4 min in the absence of PEP before adding it in the assay mixture which was composed of 50 mM Tris-HCl, pH 7.4, 20 mM KCl, 5 mM MgCl 2 , 1 mM ADP and 1.5 unit of lactate dehydrogenase.…”

Effect of murine strain on metabolic pathways of glucose production after brief or prolonged fasting

“…The primary difference between short-term and long-term fasting was, as expected, a decreased reliance on glycogenolysis for EGP. The resulting dependence on gluconeogenesis was satisfied by a decrease in the disposal of phosphoenolpyruvate through the pyruvate cycling pathway rather than an increased flux through PEPCK, suggesting an important role for pyruvate kinase in the regulation of gluconeogenesis (15,23). Another striking observation of this work is that gluconeogenesis has a much greater contribution from glycerol in mice compared with humans.…”

The role of purinergic signaling in the liver and in transplantation: effects of extracellular nucleotides on hepatic graft vascular injury, rejection and metabolism

“…Glycogen synthase is inhibited, adenosine 3': 5'-cyclic monophosphate levels are lowered and activation of phospholipase D is decreased (81,(154)(155)(156). In addition, administered ATP appears to facilitate up-regulation of the gluconeogenic enzyme, phosphoenolpyruvate carboxykinase, and down-regulation of the glycolytic enzyme, pyruvate kinase; hence promoting glucose output by the liver (157,158). Of the P2Y receptors that mainly control metabolism and proliferation, P2Y2 seems to primarily control both glycogen metabolism and proliferation-associated responses such as increased [Ca ( 2+ )](c) and modulation of mitogen-activated protein kinase cascades (82).…”