1 Unitat de Bioquímica, Campus de Bellvitge, Universitat de Barcelona, 08907 Barcelona; and 2 Department of Medical Bioanalysis, Instituto de Investigaciones Biomédicas de Barcelona, Consejo Superior de Investigaciones Científicas, Institut d'Investigacions Biomediques August Pi i Sunyer, 08036 Barcelona, Spain
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We evaluated the possibility that ischemic preconditioning could modify hepatic energy metabolism during ischemia. Accordingly, high-energy nucleotides and their degradation products, glycogen and glycolytic intermediates and regulatory metabolites, were compared between preconditioned and nonpreconditioned livers. Preconditioning preserved to a greater extent ATP, adenine nucleotide pool, and adenylate energy charge; the accumulation of adenine nucleosides and bases was much lower in preconditioned livers, thus reflecting slower adenine nucleotide degradation. These effects were associated with a decrease in glycogen depletion and reduced accumulation of hexose 6-phosphates and lactate. 6-Phosphofructo-2-kinase decreased in both groups, reducing the availability of fructose-2,6-bisphosphate. Preconditioning sustained metabolite concentration at higher levels although this was not correlated with an increased glycolytic rate, suggesting that adenine nucleotides and cAMP may play the main role in the modulation of glycolytic pathway. Preconditioning attenuated the rise in cAMP and limited the accumulation of hexose 6-phosphates and lactate, probably by reducing glycogen depletion. Our results suggest the induction of metabolic arrest and/or associated metabolic downregulation as energetic cost-saving mechanisms that could be induced by preconditioning.
adenosine 5'-triphosphate; glycogen; adenosine 3',5'-cyclic monophosphate; glucose; fructose
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
BRIEF EPISODES OF ISCHEMIA and reperfusion elicit organ tolerance to longer subsequent periods of ischemia. This phenomenon, known as "ischemic preconditioning," was first described in the heart over a decade ago (19). Recently, this protective effect was reported in the liver (22-24).
Although the beneficial effects of ischemic preconditioning have been described, the underlying mechanism(s) are not well established. In this sense, several theories have been suggested to explain the phenomenon. The most popular theory focuses on increased tissue production of substances such as nitric oxide and adenosine (23, 38). However, the possibility that preconditioning could alter energy metabolism has also been proposed in the heart (13), but to our knowledge no data have been reported in the liver.
Glucose metabolism by way of glycolysis is the major source of energy production during severe ischemia (14, 40). Although glycolysis is essential for cell survival during ischemia, it may also be detrimental because of the accumulation of glycolytic products such as lactate (21). Several studies have suggested that myocardial preconditioning could confer protection by preserving ATP stores and/or reducing lactate accumulation (15, 20). Intensive investigations are required to elucidate the cause of these two processes.
ATP degradation during ischemia leads to an acceleration of glycolysis, resulting in the net formation of lactate (30, 40). The rate of glycolysis is controlled by multiple steps distributed along this pathway and subject to control at discrete points. Likewise, the role of cAMP and fructose 2,6-bisphosphate (Fru-2,6-P2) as regulatory molecules of hepatic glycolysis/gluconeogenesis is well established (3, 8, 10, 25, 36).
Ischemia leads to a considerable increase in cAMP, which is an important factor in glucose metabolism (8, 39). cAMP, through the action of cAMP-dependent protein kinase, leads to the phosphorylation of key enzymes, including glycogen phosphorylase, 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase (PFK-2/FBPase-2), or L-type pyruvate kinase. This phosphorylation greatly affects the kinetic properties of the different enzymes implicated in the control of carbohydrate metabolism, thus resulting in changes in pathway flux (3, 8, 25).
Fru-2,6-P2, synthesized by PFK-2, is the most powerful allosteric activator of 6-phosphofructo-1-kinase (PFK-1), one of the key steps controlling glycolysis (8, 16, 25, 29, 36). Fru-2,6-P2 has been considered an important metabolite in the control of glycolysis during the few minutes after initiation of ischemia (1, 5). After the first minutes of ischemia, control of glycolysis is probably exerted by changes in adenine nucleotides such as the decrease in ATP or increase in AMP consequent to ATP degradation (8, 10, 36).
In the present work we have evaluated whether 1) ischemic preconditioning can reduce the ATP degradation and the lactate accumulation produced by ischemia and 2) these effects could be explained by the action of preconditioning on regulatory metabolites such as cAMP, Fru-2,6-P2, and/or adenine nucleotides that could trigger the activation of glycolysis after initiation of ischemia.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Surgical Procedure
Male rats (6 in each group) weighing between 250 and 300 g were used. All animals (including controls) were anesthetized with urethan (10 mg/kg ip) and placed in a supine position on a heating pad to maintain body temperature between 36°C and 37°C. To induce hepatic ischemia, laparotomy was performed and the blood supply to the right lobe of the liver was interrupted by placement of a bulldog clamp at the level of the hepatic artery and portal vein. Reflow was initiated by removing the clamp (23). The studies were performed in concordance with the European Union regulations for animal experiments (EC Guideline 86/60/CEE).Experimental Design
Preconditioning and hepatic metabolism during sustained ischemia. To study the effects of preconditioning on hepatic metabolism during sustained ischemia the following experimental groups were set up. Group 1 (n = 6) consisted of control animals subjected to anesthesia and laparotomy. Group 2 animals (n = 24 in 4 groups of 6) were subjected to increasing periods (10, 30, 60, and 90 min) of right lobe hepatic ischemia. Before the ischemic period (as in group 2), group 3 animals (n = 24) were subjected to preconditioning induced by 10 min of ischemia and 10 min of reperfusion. Control experiments were performed immediately after the hepatic preconditioning period, before the sustained ischemia. A control group of animals (n = 6) were subjected to anesthesia and laparotomy. A second group of animals (n = 6) were subjected to 10 min of ischemia followed by 10 min of reperfusion (preconditioning period). At the end of the protocol, liver samples were collected for the analysis of nucleotides (adenine nucleotides, adenine nucleosides, and bases), metabolites [glycogen, glucose 6-phosphate (Glu-6-P), fructose 6-phosphate (Fru-6-P), Fru-2,6-P2, lactate, and cAMP] and enzymes (PFK-1 and PFK-2).
Implication of cAMP in hepatic preconditioning. To evaluate whether preconditioning, by reducing the increase in cAMP a few minutes after the initiation of ischemia, could attenuate both the lactate accumulation observed during sustained ischemia and the ensuing hepatic injury, the following experimental groups were studied. In a first series of animals, tissue cAMP levels were determined a few minutes after initiation of ischemia. A group of animals (n = 12 in 2 groups of 6) were subjected to 10 min of ischemia with or without previous preconditioning. A second group of animals (n = 6) were subjected to 10 min of ischemia with previous administration of an inhibitor of adenylate cyclase, 9-(tetrahydro-2-furanyl)-9H-purin-6-amine (SQ-22536; RBI, Natick, MA) at an intraperitoneal dose of 300 µg/kg, 5 min before ischemia (33). A third group of animals (n = 6) were subjected to preconditioning before 10 min of ischemia with previous administration of an activator of adenylate cyclase, forskolin (100 µg/kg ip; Sigma Chemical, St. Louis, MO; Ref. 2), 5 min before preconditioning. Tissue samples were obtained after 10 min of ischemia and were processed to determine the tissue cAMP levels. In a second series of experiments, hexose 6-phosphates (Glu-6-P and Fru-6-P) and lactate were measured after the sustained ischemia and hepatic injury was evaluated after hepatic reperfusion. A group of animals (group A) was divided into 2 subgroups (n = 12, 6 in each group), animals subjected to 90 min of ischemia with or without previous preconditioning. A second group of animals (group B; n = 6) were subjected to 90 min of ischemia with SQ-22536 (300 µg/kg ip) 5 min before ischemia (33). A third group of animals (group C; n = 6) were subjected to 90 min of ischemia with previous preconditioning and pretreatment with forskolin (100 µg/kg ip) (2), 5 min before preconditioning. Liver samples were obtained after 90 min of ischemia to analyze hexose 6-phosphates and lactate. To evaluate the degree of hepatic injury, animals subjected to the same experimental procedures as in groups A, B, and C were subjected to 90 min of reperfusion after 90 min of ischemia. Blood samples were obtained after hepatic reperfusion and processed to determine plasma aminotransferases.
Biochemical Determinations
Nucleotide analysis. Livers were freeze-clamped, and ATP was extracted with H2O-acetone (1:1.2 vol/vol). ATP content was measured by enzymatic methods using hexokinase and glyceraldehyde-3-phosphate-dehydrogenase (34). To analyze adenine nucleotide content (ATP + ADP + AMP), adenine nucleosides (adenosine + inosine), and bases (xanthine + hypoxanthine), the livers were freeze-clamped and immediately homogenized in 10 vols of 3.6% HClO4. After homogenization, tissues were allowed to extract for 30 min at 0.5°C and were centrifuged at 850 g for 15 min. Supernatants were adjusted to pH 6.0-6.5 and centrifuged at 14,000 rpm, and then 50 µl of the supernatant were injected into Waters 717 plus Autosampler liquid chromatographic equipment (Waters, Milford, MA). Nucleotide profiles were obtained using a reversed-phase Spherisorb ODS column (C18, 5-µm particle size, 15 × 0.4 cm; Teknokroma, San Cugat, Spain) coupled to a 600 HPLC system equipped with a Waters 996 photodiode array detector. Absorbance was monitored at 254 nm. Nucleotide separation was allowed to proceed in a isocratic fashion with 100 mM ammonium phosphate (pH 5.5) until ATP, ADP, hypoxanthine, xanthine, and AMP were separated. At this point, the eluent was changed to water-methanol (96:4 vol/vol) to eluate inosine and then changed to water-methanol (60:40) to elute adenosine (11). Calibration chromatograms for the standards ATP, ADP, AMP, adenosine, inosine, hypoxanthine, and xanthine were generated by injecting 50 µl of a mixture of known concentrations. The profiles were processed by a Millennium32 system.
Liver metabolite assays. Glycogen content was measured in freeze-clamped livers. Glycogen was isolated from solubilized tissue samples by ethanol precipitation. Afterwards, glycogen was hydrolyzed with 5 N H2SO4 to glucose (27), which was measured using a commercial glucose kit from Boehringer Mannheim (Munich, Germany). Glycolysis intermediates (Glu-6-P and Fru-6-P) were extracted in 10 vols of 6% HClO4. Extracts were neutralized and then centrifuged at 5,000 g for 10 min. These metabolites were assayed fluorimetrically using glucose 6-phosphate dehydrogenase and phosphoglucose isomerase according to the method described by Lang and Michal (18). For Fru-2,6-P2 analysis, the frozen livers were homogenized in 10 vols of 50 mM NaOH. The homogenates were heated at 80°C over 10 min, cooled, and adjusted to a pH 6-7. After centrifugation at 15,000 g for 10 min, Fru-2,6-P2 was quantified in the supernatants as described by Van Schaftingen et al. (37). Lactate was extracted with H2O-acetone (1:1.2 vol/vol). The lactate content was measured using a commercial kit from Boehringer Mannheim. cAMP was extracted and quantified radioimmunologically according to the manufacturer's protocol [cyclic AMP (3H) assay system, Amersham].
Enzyme assays. PFK-1 was extracted from frozen livers in 10 vols of ice-cold 50 mM HEPES, 100 mM KCl, and 15 mM EGTA (pH 7.1) and centrifuged at 40,000 g for 10 min at 4°C. PFK-1 activity was determined by the method described by Staal et al. (31). PFK-2 activity was determined by a modification of the method described by Bartrons et al. (3). Liver samples were homogenized in 10 vols of ice-cold 20 mM Pi, 100 mM KF, 10 mM EDTA, 1 mM dithiothreitol (DTT), and 3% polyethylene glycol 6000 (pH 7.1) and centrifuged at 40,000 g for 10 min at 4°C. The supernatants were incubated at 30°C in 50 mM HEPES buffer (pH 7.1) containing (in mM) 50 KCl, 5 MgATP, 5 Fru-6-P, 17.5 Glu-6-P, 2 MgCl2, 1 DTT, and 1 Pi.
Aminotransferase determinations. The evaluation of hepatic injury was performed by enzymatic determinations of aspartate aminotransferase and alanine aminotransferase plasma levels using a commercial kit from Boehringer Mannheim.
Statistics
Experimental results are expressed as means ± SE. Means of different groups were compared using a one-way ANOVA. Student's t-test was performed for evaluation of significant differences between groups. Significance was determined at the 5% level (P < 0.5). ![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Effect of Preconditioning on Adenine Nucleotides During Sustained Ischemia
To control cell energy status, adenine nucleotides were measured in all groups of animals. As shown in Fig. 1, in both preconditioned and nonpreconditioned groups ATP levels decreased as a function of the length of the ischemic period. However, ATP levels were maintained markedly higher in preconditioned livers. Adenine nucleotides (ATP + ADP + AMP), adenine nucleosides (adenosine + inosine), and bases (hypoxanthine + xanthine) are presented in Fig. 1. Adenine nucleotides in nonpreconditioned livers showed decreasing levels as a function of the length of the ischemic period, and the corresponding adenylate energy charge (ATP + [1/2]ADP/ATP + ADP + AMP) was reduced from 0.80 ± 0.02 to 0.28 ± 0.03 at 30 min as a consequence of the decrease in ATP and the increase in AMP concentrations. However, preconditioning caused a slowing in the decrease of adenine nucleotides and adenylate energy charge, because this parameter was reduced in this case from 0.80 ± 0.02 to 0.63 ± 0.02. The accumulation of nucleosides and bases found after ischemia was significantly slower when ischemia was preceded by preconditioning (Fig. 1).
|
Effect of Preconditioning on Hepatic Metabolites and Enzymes During Sustained Ischemia
Glycogen concentration decreased as a function of the length of the ischemic period. However, glycogen levels were always higher in the preconditioned livers (Fig. 2). To ascertain whether the decrease in glycogen had an immediate effect on the flux through the glycolytic/gluconeogenic pathway, lactate and hexose 6-phosphate concentrations were measured. As shown in Fig. 2, lactate concentration increased progressively in nonpreconditioned livers, achieving a maximum level at 30 min and remaining stable for the rest of the measured time. In contrast, when preconditioning was carried out the lactate levels throughout the ischemic period were significantly reduced. Glu-6-P and Fru-6-P also increased after ischemia, achieving a maximal concentration at 30 min and remaining high up to 90 min. However, when preconditioning preceded ischemia, Glu-6-P and Fru-6-P levels were similar to those found in the control group. Because PFK-1 catalyzes one of the regulatory reactions of glycolytic pathway, we have measured this enzymatic activity in preconditioned and nonpreconditioned livers. The results obtained did not show significant variations (data not shown).
|
Among the numerous effectors of liver PFK-1, Fru-2,6-P2 is
the most powerful activator. Its concentration decreased in both groups, reaching lowest values at 30 min of ischemia. However, the decrease was always more modest in the preconditioned group (Fig.
3). To study the mechanism by which
ischemia lowers Fru-2,6-P2 concentration, we measured the
enzyme responsible for its synthesis. PFK-2 activity was markedly
reduced in both groups (Fig. 3), although the decrease was attenuated
in the preconditioned group. The activity of this multimodulated enzyme
depends on its substrates (Fru-6-P and ATP), modulators (AMP,
Pi, citrate, glycerol-3-P), and also cAMP, because PFK-2 is
mainly regulated by cAMP-dependent protein kinase. cAMP, through
cAMP-dependent protein kinase, leads to phosphorylation of PFK-2, thus
reducing its activity (3, 8, 25,
29, 36). As shown in Fig. 3, ischemia leads
initially to a significant increase in cAMP concentration, which
remains high up to 30 min. When preconditioning preceded ischemia
smaller changes in cAMP were observed. The preconditioning period did not significantly modify any metabolic parameter analyzed compared with
the control group (data not shown).
|
Role of cAMP in Hepatic Preconditioning
As shown in Fig. 4, preconditioning attenuated the increase in cAMP found a few minutes after the initiation of sustained ischemia (10 min). To ascertain the role of cAMP, we studied the effects of an inhibitor of adenylate cyclase (SQ-22536) and an activator of adenylate cyclase (forskolin) in the nonpreconditioned and preconditioned groups, respectively. SQ-22536 at a dose of 300 µg/kg prevented the increase in cAMP, whereas forskolin at a dose of 100 µg/kg produced metabolite levels similar to those obtained after ischemia. The differences in cAMP found after initiation of ischemia (10 min) between the different groups of the study were reflected in changes in the accumulation of hexose 6-phosphate and lactate at the end of sustained ischemia (90 min) and hepatic injury (evaluated by transaminase levels in plasma) after 90 min of reperfusion, as illustrated in Fig. 4.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The liver is damaged by ischemia in liver transplantation or surgical procedures, and reperfusion after ischemia results in functional impairment. Prolonged ischemia causes massive necrosis and induces liver failure (26). Metabolic and functional tolerance to ischemia can be obtained by interventions that limit anaerobic glycolysis and consequent production of lactate (21). In addition to limiting injury, interventions that prevent the loss of critical metabolites such as ATP could also be effective (35).
Our recent studies in liver have shown the effectiveness of ischemic preconditioning induced by 10 min of ischemia followed by 10 min of reperfusion before a sustained ischemia of 90 min (22-24). However, the possibility that preconditioning could modify hepatic energy metabolism had not been determined as we have done in the present study, in which we evaluated the effect of preconditioning on energy metabolism throughout the ischemic period (10, 30, 60, and 90 min).
The results presented here confirm the well-established effect of anoxia on liver adenine nucleotides (7, 10, 35) and show that ATP was nearly lost as a result of the ischemic state, in which ATP is quickly hydrolyzed to ADP and not replaced, owing to the lack of oxygen supply. As expected, the decrease in ATP levels a few minutes after the initiation of ischemia was associated with the increase in AMP levels (Fig. 1). The entire pool of adenine nucleotides and the liver energy charge decreased by ~55% and ~65%, respectively. Preconditioning preserved more of the ATP, adenine nucleotide pool, and adenylate energy charge (decreased by ~20%) during sustained ischemia. In contrast, AMP was significantly less increased in preconditioned livers. Also, the accumulation of adenine nucleosides and bases was much lower in preconditioned livers, reflecting slower adenine nucleotide degradation.
The mechanisms by which preconditioning leads to ATP preservation are not well established. A study in dog hearts indicated that ATP preservation could be related to decreased ATP utilization. Preconditioning resulted in higher ATP levels with a concomitant decrease in lactate production (21). However, other studies in rabbit hearts indicated that ATP preservation could be related to increased ATP production from anaerobic glycolysis. Preconditioning resulted in higher ATP levels and also increased myocardial lactate production during the ischemic process (12). In the liver, tissue ATP content was markedly higher in the preconditioned group (Fig. 1). However, ATP preservation does not seem to be related to ATP production via anaerobic glycolysis because there is a close inverse relationship between ATP and glycolytic activity, as estimated by lactate production (Fig. 2). Consequently, ATP preservation probably results from decreased ATP utilization. For instance, this could also explain the slower ATP degradation in preconditioned livers (Fig. 1).
As shown in Fig. 2, preconditioning substantially slowed lactate accumulation measured during the 90-min ischemia. Furthermore, the final extent of lactate accumulation was less. This indicates that the glycolytic rates were substantially slower in preconditioned livers. ATP degradation after ischemia leads to the activation of glycolysis, resulting in the net formation of lactate (30, 40). In liver, the increases in cAMP and Fru-2,6-P2 as a consequence of ischemia are signals that trigger the activation of glycolysis. This effect is mediated by the action of these metabolites on main enzymes of the glycolytic pathway (8, 25, 36, 39). Therefore, the possibility that preconditioning could reduce the increase in the regulatory molecules of glycolytic pathway, thus attenuating the activation of glycolysis and consequently the accumulation of lactate after ischemia, could be considered.
The different cAMP levels observed between preconditioned and nonpreconditioned livers (Fig. 3) could explain, in part, the differences in glycolytic metabolism. It has been reported that the increase in cAMP levels after initiation of ischemia leads to the activation of glycogen phosphorylase via cAMP-dependent protein kinase. This initiates glycogenolysis and causes the accumulation of hexose 6-phosphates that proceed down the glycolytic pathway to form lactate (8, 32). We evaluated whether preconditioning by reducing the increase in cAMP a few minutes after the initiation of ischemia could induce changes in hepatic metabolism during the sustained ischemia. For this purpose, we tried to modify the cAMP levels in both preconditioned and nonpreconditioned groups by activation or inhibition of adenylate cyclase, respectively. As shown in Fig. 4, the attenuation of the increase of cAMP after ischemia by the administration of an inhibitor of adenylate cyclase reduced the accumulation of hexose 6-phosphates and lactate after ischemia. This also resulted in reduced transaminase levels after hepatic reperfusion (Fig. 4). However, the increase in cAMP in the preconditioned group induced by the stimulation of adenylate cyclase abolished the effects of preconditioning on glycolytic metabolism and hepatic injury. Hexose 6-phosphate, lactate, and transaminase levels similar to those found in the nonpreconditioned group were found. These results suggest that preconditioning, by way of an attenuation in the increase in cAMP levels after the initiation of ischemia, limited the availability of hexose 6-phosphates for anaerobic glycolysis, probably by decreased glycogenolysis through cAMP-dependent protein kinase. This resulted in reduced lactate accumulation during the ischemia, thus attenuating the hepatic injury associated with this process.
One of the glycolytic regulatory enzymes is PFK-1, which
catalyzes the formation of Fru-1,6-P2. The activity of
PFK-1 was not significantly changed in preconditioned or
nonpreconditioned livers, although its regulation could be a
consequence of changes in the concentration of their allosteric
modulators. Among the numerous effectors of PFK-1,
Fru-2,6-P2 is its most potent stimulator (8,
25, 29, 36). Some studies have
reported that Fru-2,6-P2 can trigger the activation of
glycolysis a few minutes after initiation of ischemia, decreasing after
5 min (1, 5). The fall in Fru-2,6-P2 during sustained ischemia (Fig. 3) was
associated with decreased PFK-2 activity. As reported in the literature
(3, 5, 8, 25,
29, 36), the reduced PFK-2 activity during ischemia could occur as a consequence of the phosphorylation of PFK-2
through cAMP-dependent protein kinase because the cAMP concentrations are increased (see Fig. 5). In line with
these data, when preconditioning preceded ischemia, the higher
Fru-2,6-P2 levels were associated with a lesser decrease in
PFK-2 activity and reduced cAMP levels (Fig. 3). It should be noted
that the Fru-2,6-P2 remaining in anoxic liver, and
especially in preconditioned liver, might still exert a stimulatory
effect. However, the fact that glycolysis was decreased in
preconditioned livers, which showed higher Fru-2,6-P2 levels, suggests that this bisphosphorylated metabolite is not crucial
in the control of the glycolytic pathway. We and others (8, 10, 36) suggest that when
glycolysis is the only source of ATP, such as during anoxia, changes in
adenine nucleotides such as decrease in ATP or increase in AMP probably
play the main regulatory role in the control of glycolytic pathway, at
least after the first minutes of ischemia (see Fig. 5).
|
Although some degree of ischemia and hypoxia can be sustained by all animals, both conditions are incompatible with survival of most mammalian tissues. The demand for glucose or glycogen for anaerobic glycolysis may rise drastically as a means of making up for the energetic shortfall. This approach, however, leads to enormous expenditure of substrates and to high accumulation rates of metabolic waste products. Survival time of cells during ischemia is, in general, directly related to the degree of metabolic depression achieved (6, 9). The energetic cost savings realized by the organism is a consequence primarily of the ability to depress the ion pumping activities of cells, macromolecular synthesis, and turnover. In hypoxia-tolerant animals these problems are resolved through two mechanisms, 1) metabolic arrest (reduced or unchanging glycolytic flux at reduced O2 availability) and 2) stabilized membrane functions (4, 6, 9, 17, 28).
Together, the results of the present study allow us to suggest a mechanism to explain the effects of preconditioning on hepatic metabolism during sustained ischemia, as summarized in Fig. 5. The reduced ATP degradation induced by preconditioning, resulting probably from decreased ATP utilization, would attenuate the net formation of lactate. This effect could be mediated by the action of preconditioning on regulatory metabolites such as cAMP and AMP. Thus the reduced cAMP levels induced by preconditioning could attenuate the modification of key enzymes of the glycogenolytic and glycolytic pathway caused by cAMP-dependent protein kinase. This, in addition to the reduced AMP levels induced by preconditioning, could reduce the activity of key enzymes and the availability of intermediates for anaerobic glycolysis, thus attenuating the glycolytic rate, decreasing the accumulation of acid waste products, and allowing cells to survive in these adverse conditions. The results reported here point to the induction of metabolic arrest and/or associated metabolic downregulation as the energetic cost saving mechanisms that could be induced by preconditioning.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank E. Adanero and N. Goren for technical assistance.
![]() |
FOOTNOTES |
---|
This study was supported by the Fondo de Investigaciones Sanitarias (FIS) through Project 00/0038-1 and by Dirección General de Investigación Científica y Técnica (PM 97/0114). A. Manzano received a research fellowship from Ministerio de Educación y Ciencia and L. Riera from Fundació Pi i Sunyer.
Address for reprint requests and other correspondence: J. Roselló-Catafau, Dept. of Medical Bioanalysis, Instituto de Investigaciones Biomédicas de Barcelona, CSIC-IDIBAPS, C/ Rosellón, 161, 6a y 7a planta 08036-Barcelona, Spain (E-mail: jrcbam{at}iibb.csic.es).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Received 10 September 1999; accepted in final form 12 February 2000.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Ambrosio, S,
Ventura F,
Rosa JL,
and
Bartrons R.
Fructose 2,6 bisphosphate in hypoglycemic rat brain.
J Neurochem
57:
200-203,
1991[ISI][Medline].
2.
Avedano, CE,
and
Billman GE.
Effect of interventions that increase cyclic AMP levels on susceptibility to ventricular fibrillation in unanesthetized dogs.
Eur J Pharmacol
255:
1-3,
1994[ISI][Medline].
3.
Bartrons, R,
Hue L,
Van Schaftingen E,
and
Hers HG.
Hormonal control of fructose 2,6-bisphosphate concentration in isolated rat hepatocytes.
Biochem J
136:
115-124,
1983.
4.
Dzeja, PP,
and
Terzic A.
Phosphotransfer reactions in the regulation of ATP-sensitive K+ channels.
FASEB J
12:
523-529,
1988
5.
Federov, S,
and
Uyeda K.
Oscillation in fructose 2,6-bisphosphate levels and in the phosphorylation states of fructose 6-phosphate, 2-kinase: fructose-2,6-bisphosphatase in ischemic rat liver.
J Biol Chem
267:
20826-20830,
1992
6.
Hand, SC,
and
Hardewig I.
Downregulation of cellular metabolism during environmental stress: mechanisms and implications.
Annu Rev Physiol
58:
539-563,
1996[ISI][Medline].
7.
Hems, DA,
and
Brosnan JR.
Effects of ischaemia on content of metabolites in rat liver and kidney in vivo.
Biochem J
120:
105-111,
1970[ISI][Medline].
8.
Hers, HG,
and
Hue L.
Gluconeogenesis and related aspects of glycolysis.
Annu Rev Biochem
52:
617-653,
1983[ISI][Medline].
9.
Hochachka, PW.
Defense strategies against hypoxia and hypothermia.
Science
231:
234-241,
1986[ISI][Medline].
10.
Hue, L,
and
Rider MH.
Role of fructose 2,6-bisphosphate in the control of glycolysis in mammalian tissues.
Biochem J
245:
313-324,
1987[ISI][Medline].
11.
Huel-Ride, EA,
Lewis WR,
Veronee CD,
and
Lowe JE.
Simple step gradient elution of the major high-energy compounds and their catabolites in cardiac muscle using high-performance liquid chromatography.
J Chromatogr Biomed Appl
377:
165-174,
1986.
12.
Janier, MF,
Vanoverschelde JLJ,
and
Bergmann SR.
Ischemic preconditioning stimulates anaerobic glycolysis in the isolated rabbit heart.
Am J Physiol Heart Circ Physiol
267:
H1353-H1360,
1994
13.
Jennings, RB,
Murry CE,
and
Reimer KA.
Energy metabolism in preconditioned and control myocardium: effect of total ischemia.
J Mol Cell Cardiol
23:
1449-1458,
1991[ISI][Medline].
14.
Jennings, RB,
Reimer KA,
Steenbergen C,
and
Schaper J.
Total ischemia. III. Effect of inhibition of anaerobic glycolysis.
J Mol Cell Cardiol
21:
37-54,
1989[ISI][Medline].
15.
Kida, M,
Fujiwara H,
Ishida M,
Kawai C,
Ohura M,
Miura I,
and
Yabuuchi Y.
Ischemic preconditioning preserves creatine phosphate and intracellular pH.
Circulation
84:
2495-2503,
1991[Abstract].
16.
Krebs, HA.
The Pasteur effect and the relations between respiration and fermentation.
Essays Biochem
8:
1-34,
1972[Medline].
17.
Krumschnabel, G,
Biasi C,
Schwarzbaum PJ,
and
Wieser W.
Membrane-metabolic coupling and ion homeostasis in anoxia-tolerant and anoxia-intolerant hepatocytes.
Am J Physiol Regulatory Integrative Comp Physiol
270:
R614-R620,
1996
18.
Lang, G,
and
Michal G.
D-Glucose-6-phosphate and D-fructose-6-phosphate.
In: Methods of Enzymatic Analysis, edited by Bergmeyer HU.. New York: Academic, 1974, vol. VI, p. 1238.
19.
Murry, CE,
Jenning RB,
and
Reimer KA.
Preconditioning with ischemia: a delay in lethal cell injury in ischemic myocardium.
Circulation
74:
1124-1136,
1986[Abstract].
20.
Murry, CE,
Richard VJ,
Reimer KA,
and
Jennings RB.
Ischaemic preconditioning slows energy metabolism and delays ultrastructural damage during a sustained ischemic episode.
Circ Res
66:
913-931,
1990[Abstract].
21.
Neely, RR,
and
Grotyohann LW.
Role of glycolytic products in damage to ischemic myocardium. Dissociation of adenosine triphosphate levels and recovery of function of reperfused ischemic hearts.
Circ Res
55:
816-824,
1984[Abstract].
22.
Peralta, C,
Closa D,
Hotter G,
Gelpí E,
Prats N,
and
Roselló-Catafau J.
Liver ischemic preconditioning is mediated by the inhibitory action of nitric oxide on endothelin.
Biochem Biophys Res Commun
229:
264-270,
1996[ISI][Medline].
23.
Peralta, C,
Hotter G,
Closa D,
Gelpí E,
Bulbena O,
and
Rosselló-Catafau J.
Protective effect of preconditioning on the injury associated to hepatic ischemia-reperfusion in the rat: role of nitric oxide and adenosine.
Hepatology
25:
934-937,
1997[ISI][Medline].
24.
Peralta, C,
Hotter G,
Closa D,
Prats N,
Xaus C,
Gelpí E,
and
Roselló-Catafau J.
The protective role of adenosine in inducing nitric oxide synthesis in rat liver ischemia preconditioning is mediated by activation of adenosine A2 receptors.
Hepatology
29:
126-132,
1999[ISI][Medline].
25.
Pilkis, SJ,
Claus TH,
Kurland Y,
and
Lange AJ.
6-Phosphofructo-2-kinase/fructose 2,6-bisphosphatase: a metabolic signaling enzyme.
Annu Rev Biochem
64:
799-785,
1995[ISI][Medline].
26.
Popper, H.
Hepatocellular degeneration and death.
In: The Liver: Biology and Pathobiology, edited by Arias I,
Popper H,
Schachter D,
and Shafritz DA.. New York: Raven, 1982, p. 771.
27.
Roe, JH,
and
Dailey RE.
Determination of glycogen with the anthrone reagent.
Anal Biochem
15:
245-250,
1996.
28.
Roig, T,
Bartrons R,
and
Bermúdez J.
Exogenous fructose 1,6-bisphosphate reduces K+ permeability in isolated rat hepatocytes.
Am J Physiol Cell Physiol
273:
C473-C478,
1997
29.
Rousseau, GG,
and
Hue L.
Mammalian 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase: a bifunctional enzyme that controls glycolysis.
Prog Nucleic Acid Res Mol Biol
45:
99-127,
1993[ISI][Medline].
30.
Rovetto, MJ,
Lamberton WF,
and
Neely JR.
Mechanisms of glycolytic inhibition in ischemic rat hearts.
Circ Res
37:
742-751,
1975[Abstract].
31.
Staal, GEJ,
Kalff A,
Heesbeen EC,
Van Veelen CWM,
and
Rijksen G.
Subunit composition, regulatory properties, and phosphorylation of phosphofructokinase from human gliomas.
Cancer Res
47:
5047-5051,
1987[Abstract].
32.
Stantley, WC,
Lopaschuk GD,
Hall JL,
and
McCormack JG.
Regulation of myocardial carbohydrate metabolism under normal and ischaemic conditions. Potential for pharmacological interventions.
Cardiovasc Res
33:
243-257,
1997[ISI][Medline].
33.
Tolliver, BK,
Ho LB,
Fox LM,
and
Berger SP.
Necessary role for ventral tegmental area adenylate cyclase and protein kinase A in induction of behavioral sensitization to intraventral tegmental area amphetamine.
J Pharmacol Exp Ther
289:
38-47,
1999
34.
Trautschold, I,
Lamprecht W,
and
Schweitzer G.
ATP UV-method with hexoquinase and glucose-6-phosphate dehydrogenase.
In: Methods of Enzymatic Analysis, edited by Bergmeyer HU.. New York: Academic, 1974, vol. VII, p. 347.
35.
Vanoverschelde, JLJ,
Janier MF,
Bakke JE,
Marshall DR,
and
Bergmann SR.
Rate of glycolysis during ischemia determines extent of ischemic injury and functional recovery after reperfusion.
Am J Physiol Heart Circ Physiol
267:
H1785-H1794,
1994
36.
Van Schaftingen, E.
Fructose 2,6-bisphosphate.
Adv Enzymol
59:
315-395,
1987[ISI][Medline].
37.
Van Schaftingen, E,
Lederer B,
Bartrons R,
and
Hers HG.
A kinetic study of pyrophosphate: fructose-6-phosphate phosphotransferase from potato tubers.
Eur J Biochem
129:
191-195,
1982[Abstract].
38.
Vegh, A,
Szekeres L,
and
Parrat JR.
Preconditioning of the ischemic myocardium; involvement of the L-arginine nitric oxide pathway.
Br J Pharmacol
107:
648-652,
1992[Abstract].
39.
Watanabe, H,
and
Ishii S.
The effect of brain ischemia of the levels of cyclic AMP and glycogen metabolism in gerbil brain in vivo.
Brain Res
102:
385-389,
1976[ISI][Medline].
40.
Woods, HF,
and
Krebs HA.
Lactate production in the perfused rat liver.
Biochem J
125:
129-139,
1971[ISI][Medline].