Increased Lactate Accumulation Research Articles

In VitroInfluences between Pancreatic Adenocarcinoma Cells and Pancreatic Islets

Background.Interactions have been found between exocrine pancreatic adenocarcinoma and islets of Langerhans. Growth of pancreatic adenocarcinoma cells can be regulated by islet hormones such as insulin and somatostatin. Conversely, dysfunction of endocrine pancreas frequently accompanies the exocrine malignancy. The mechanisms underlying these interactions have not been defined.Materials and methods.Human pancreatic adenocarcinoma cells (HPAF cells) were cocultured with isolated rat pancreatic islets in two-compartment wells. HPAF cells and islets cultured in separate wells served as controls. In separate experiments, HPAF cells were incubated with two concentrations of exogenous insulin, including one reflecting the levels of insulin secretion seen in the coculture experiments.Results.Proliferation of HPAF cells was increased by about 50% following a 2- or 5-day incubation with pancreatic islets (P< 0.05). Coculture of HPAF cells and pancreatic islets was associated with a greater reduction in glucose concentrations (P< 0.01) and an increase in lactate accumulation (P< 0.05) in the culture media. Insulin concentrations in the media were significantly decreased during the first 2–3 days of the coculture incubation (P< 0.05). In contrast, insulin secretion from control islets was not significantly decreased until the fifth day of the experiment. The growth of HPAF cells was stimulated by both concentrations of exogenous insulin (P< 0.05). The insulin-stimulated HPAF cells also showed an enhanced glucose consumption and lactate production (P< 0.05).Conclusions.Pancreatic islets regulate both growth and glucose metabolism of adjacent exocrine cancer cells. β-cell-derived insulin may be one of the factors inducing these effects. Insulin release from islet β-cells is compromised in the presence of exocrine cancer cells.

Adenosine, inosine, and guanosine protect glial cells during glucose deprivation and mitochondrial inhibition: correlation between protection and ATP preservation.

The purpose of this study was to determine the mechanism by which adenosine, inosine, and guanosine delay cell death in glial cells (ROC-1) that are subjected to glucose deprivation and mitochondrial respiratory chain inhibition with amobarbital (GDMI). ROC-1 cells are hybrid cells formed by fusion of a rat oligodendrocyte and a rat C6 glioma cell. Under GDMI, ATP was depleted rapidly from ROC-1 cells, followed on a much larger time scale by a loss of cell viability. Restoration of ATP synthesis during this interlude between ATP depletion and cell death prevented further loss of viability. Moreover, the addition of adenosine, inosine, or guanosine immediately before the amobarbital retarded the decline in ATP and preserved cell viability. The protective effects on ATP and viability were dependent on nucleoside concentration between 50 and 1,500 microM. Furthermore, protection required nucleoside transport into the cell and the continued presence of nucleoside during GDMI. A significant positive correlation between ATP content at 16 min and cell viability at 350 min after the onset of GDMI was established (r = 0.98). Modest increases in cellular lactate levels were observed during GDMI (1.2 nmol/mg/min lactate produced); however, incubation with 1,500 microM inosine or guanosine increased lactate accumulation sixfold. The protective effects of inosine and guanosine on cell viability and ATP were >90% blocked after treatment with 50 microM BCX-34, a nucleoside phosphorylase inhibitor. Accordingly, lactate levels also were lower in BCX-34-treated cells incubated with inosine or guanosine. We conclude that under GDMI, the ribose moiety of inosine and guanosine is converted to phosphorylated glycolytic intermediates via the pentose phosphate pathway, and its subsequent catabolism in glycolysis provides the ATP necessary for maintaining plasmalemmal integrity.

Preconditioning human cardiomyocytes and endothelial cells

Background: The effects of simulated “ischemia” and “reperfusion” were evaluated in cell cultures of human ventricular cardiomyocytes and human saphenous vein endothelial cells. Methods: Myocyte and endothelial cell cultures were exposed to a low volume (1.5 ml) of either hypoxic (oxygen tension = 16 mm Hg) or anoxic (oxygen tension = 0 mm Hg) phosphate-buffered saline solution for 90 minutes (“ischemia”) followed by 30 minutes of simulated “reperfusion.” Cell injury was evaluated by trypan blue exclusion. Next, the effects of a preconditioning stimulus were evaluated by a brief (10 minute) exposure to hypoxic or anoxic ischemia and 10 minutes of reperfusion before prolonged (90 minutes) anoxic ischemia. Finally, the effects of anoxic preconditioning on intracellular lactate accumulation and extracellular lactate and acid release were assessed. Results: “Ischemia” and “reperfusion” resulted in greater injury to endothelial cells than to cardiomyocytes. In both cell types, anoxic ischemia resulted in greater injury than hypoxic ischemia. Preconditioning reduced cell injury in myocytes but not in endothelial cells. Endothelial cells produced more lactate than cardiomyocytes under normoxic conditions. Ischemia increased lactate accumulation and release in cardiomyocytes but not endothelial cells. Preconditioning reduced lactate accumulation and release in cardiomyocytes but not endothelial cells. Conclusions: Endothelial cells were more susceptible to the same period of simulated ischemia than cardiomyocytes. Preconditioning protected cardiomyocytes but not endothelial cells from a subsequent prolonged period of ischemia and reperfusion. (J Thorac Cardiovasc Surg 1998;115:210–9)

Isoflurane Preserves Adenosine Triphosphate Levels in Anoxic Isolated Rat Hepatocytes by Stimulating Glycolytic Adenosine Triphosphate Formation

The hypothesis that general anesthetics protect energy reserves by decreasing energy demand is widely accepted but poorly substantiated. Isoflurane at clinical doses preserved adenosine triphosphate (ATP) levels in anoxic isolated hepatocytes. Specific inhibitors were used to block mitochondrial and/or glycolytic ATP formation to ascertain whether pathways of energy supply or demand, or both, were involved in ATP preservation by isoflurane. Hepatocytes were isolated from fed adult male rats after perfusing livers with Krebs buffer containing collagenase. Cells were incubated in Krebs buffer for 0-30 min at 25 degrees C under N2/CO2 (95%/5%) +/- isoflurane 0.63 mM in liquid phase. Oligomycin, iodoacetate, or fasting were used to block mitochondrial and glycolytic ATP formation. Under anoxia alone, ATP levels declined more slowly in the presence than in the absence of isoflurane, confirming the ATP-protective effect of isoflurane reported previously. With oligomycin plus iodoacetate blocking all ATP formation, ATP decline (representing pure ATP consumption) was not slowed by isoflurane. Isoflurane's protective effect recurred when glycolytic ATP supply was restored by incubating with oligomycin only. The protective effect was accompanied by increased lactate accumulation, and both effects-ATP preservation and lactate formation-were similarly dependent on isoflurane concentration. We conclude that the protective effect of isoflurane on energy status in anoxic isolated hepatocytes was not associated with reduced ATP demand but with enhanced ATP supply via stimulation of glycolysis.

Isoflurane Preserves Adenosine Triphosphate Levels in Anoxic Isolated Rat Hepatocytes by Stimulating Glycolytic Adenosine Triphosphate Formation

The hypothesis that general anesthetics protect energy reserves by decreasing energy demand is widely accepted but poorly substantiated.Isoflurane at clinical doses preserved adenosine triphosphate (ATP) levels in anoxic isolated hepatocytes. Specific inhibitors were used to block mitochondrial and/or glycolytic ATP formation to ascertain whether pathways of energy supply or demand, or both, were involved in ATP preservation by isoflurane. Hepatocytes were isolated from fed adult male rats after perfusing livers with Krebs buffer containing collagenase. Cells were incubated in Krebs buffer for 0-30 min at 25 degrees C under N2/CO2 (95%/5%) +/- isoflurane 0.63 mM in liquid phase. Oligomycin, iodoacetate, or fasting were used to block mitochondrial and glycolytic ATP formation. Under anoxia alone, ATP levels declined more slowly in the presence than in the absence of isoflurane, confirming the ATP-protective effect of isoflurane reported previously. With oligomycin plus iodoacetate blocking all ATP formation, ATP decline (representing pure ATP consumption) was not slowed by isoflurane. Isoflurane's protective effect recurred when glycolytic ATP supply was restored by incubating with oligomycin only. The protective effect was accompanied by increased lactate accumulation, and both effects--ATP preservation and lactate formation--were similarly dependent on isoflurane concentration. We conclude that the protective effect of isoflurane on energy status in anoxic isolated hepatocytes was not associated with reduced ATP demand but with enhanced ATP supply via stimulation of glycolysis. (Anesth Analg 1996;82:1261-7)

Protective properties of amino acids in liver preservation: effects of glycine and a combination of amino acids on anaerobic metabolism and energetics

Background/Aims/Methods: In this study, we investigated the hepatoprotective effects of three storage solutions containing glycine (180 mM), glycylglycine (180 mM), and a mixture of 20 amino acids (combined concentration of 180 mM) on energy metabolism and levels of glucose and lactate (as an index of glycolytic flux) in rat livers. All effects were compared to those of livers flushed/stored with a modified University of Wisconsin solution. Results: Glycine-treatment showed no improvement in liver energetics (ATP, ADP, AMP) and lactate accumulation; this solution had the lowest buffering capacity of the four tested (approximately 30% of the University of Wisconsin solution). The glycylglycine solution had the highest buffering capacity of the four solutions tested (including University of Wisconsin solution). Complete titration of the glycine-, combined amino acids-, and University of Wisconsin solutions (from 8.0 to pH=6.0) resulted in a minor decrease in glycylglycine buffer pH; pH dropped by 0.2 pH units. In glycylglycine-treated livers, energetics showed an improvement over the first 1 h cold storage; ATP and ‘energy charge’ values remained high and ADP levels (and consequently total adenylate contents) were 0.7–2.4 μmol/g greater than livers stored in University of Wisconsin solution. A 2-fold increase in lactate accumulation suggested that the improvement in liver energetics for the glycylglycine buffer was due to maintained flux through glycolysis brought about by enhanced buffering capacity. The solution containing a combination of amino acids exhibited maximum maintenance of liver energetics via increased glycolytic flux, despite its slightly inferior buffering capacity (85% of University of Wisconsin solution). ATP levels were maintained over the first 2 h storage and ADP levles (and consequently, total adenylate contents) were 1.2–2.1 μmol/g greater than University of Wisconsin solution-treated livers during the entire 24 h storage period. Energy charge values for livers treated with the combination of amino acids were also significantly higher than with glycine-, glycylglycine- and University of Wisconsin solution-treatment; even at 24 h, energy charge was 0.36 (comparable to only 4 h storage in University of Wisconsin solution). Conclusions: Our data suggest that a combination of amino acids may be required for maximum protection of the liver, and furthermore there may be several independent mechanisms, including buffering capacity, responsible for cytoprotection of the liver during cold storage.

Calorie sources and recovery from central nervous system ischemia

Glucose is the primary substrate for the energy requirements of the nervous system. Nevertheless, administration of glucose to critically ill patients with central nervous system trauma may have adverse effects on their neurologic recovery. The purpose of this study was to evaluate the effects of other sources of nonprotein calories on spinal cord lactate accumulation and on electrophysiologic recovery after a period of severe spinal cord ischemia. Two randomized, blinded studies were performed: one of glycolytic energy substrates (fructose, xylitol, sorbitol, glycerol) and one of ketogenic energy substrates (beta-hydroxybutyrate, acetate, butyrate). College teaching hospital laboratory. New Zealand albino rabbits (weight 3.5 to 4.5 kg). After infusion of the randomly assigned treatment, temporary ischemia was produced in the lumbosacral spinal cord by occluding the abdominal aorta with a balloon catheter. Blood concentrations of glucose, lactate, pyruvate, and ketone bodies and spinal cord dialysate concentration of lactate were measured before and after infusion of the assigned treatment, and during ischemia and during the first 2 hrs after reperfusion. Spinal somatosensory evoked potentials were recorded during ischemia to assure a similar severity of ischemia in all animals and during the first 2 hrs after reperfusion as a measure of electrophysiologic recovery. Infusion of the glycolytic nutrients xylitol and fructose increased blood glucose and lactate concentrations, and resulted in increased lactate accumulation in the spinal cord during ischemia and resulted in a significantly poorer recovery of the spinal somatosensory evoked potential than infusion of saline. Infusion of sorbitol and glycerol did not have these adverse effects in the doses administered. None of the ketogenic nutrients increased blood glucose concentration or increased lactate accumulation in the spinal cord during ischemia when compared with infusion of saline. Infusion of butyrate and acetate caused arterial hypotension and resulted in a poorer recovery of the spinal somatosensory evoked potential than saline. Infusion of beta-hydroxybutyrate did not have an adverse effect on blood pressure or on evoked potential recovery. Glycerol, sorbitol, and beta-hydroxybutyrate deserve further evaluation as potential nonprotein calorie sources in patients with neurologic injury. Xylitol and fructose are not suitable since these substrates resulted in hyperglycemia and increased lactate accumulation in the central nervous system, and had detrimental effects on electrophysiologic recovery after ischemia. The short-chain fatty acids (acetate and butyrate) also had adverse effects on electrophysiologic recovery after ischemia, probably because of their hypotensive effects when given intravenously, rather than from the effects of their metabolism.

Calorie sources and recovery from central nervous system ischemia

Objectives: Glucose is the primary substrate for the energy requirements of the nervous system. Nevertheless, administration of glucose to critically ill patients with central nervous system trauma may have adverse effects on their neurologic recovery. The purpose of this study was to evaluate the effects of other sources of nonprotein calories on spinal cord lactate accumulation and on electrophysiologic recovery after a period of severe spinal cord ischemia. Design: Two randomized, blinded studies were performed: one of glycolytic energy substrates (fructose, xylitol, sorbitol, glycerol) and one of ketogenic energy substrates (β-hydroxy-butyrate, acetate, butyrate). Setting: College teaching hospital laboratory. Subjects: New Zealand albino rabbits (weight 3.5 to 4.5 kg). Interventions: After infusion of the randomly assigned treatment, temporary ischemia was produced in the lumbosacral spinal cord by occluding the abdominal aorta with a balloon catheter. Measurements and Main Results: Blood concentrations of glucose, lactate, pyruvate, and ketone bodies and spinal cord dialysate concentration of lactate were measured before and after infusion of the assigned treatment, and during ischemia and during the first 2 hrs after reperfusion. Spinal somatosensory evoked potentials were recorded during ischemia to assure a similar severity of ischemia in all animals and during the first 2 hrs after reperfusion as a measure of electrophysiologic recovery. Infusion of the glycolytic nutrients xylitol and fructose increased blood glucose and lactate concentrations, and resulted in increased lactate accumulation in the spinal cord during ischemia and resulted in a significantly poorer recovery of the spinal somatosensory evoked potential than infusion of saline. Infusion of sorbitol and glycerol did not have these adverse effects in the doses administered. None of the ketogenic nutrients increased blood glucose concentration or increased lactate accumulation in the spinal cord during ischemia when compared with infusion of saline. Infusion of butyrate and acetate caused arterial hypotension and resulted in a poorer recovery of the spinal somatosensory evoked potential than saline. Infusion of β-hydroxybu-tyrate did not have an adverse effect on blood pressure or on evoked potential recovery. Conclusions: Glycerol, sorbitol, and β-hydroxybutyrate deserve further evaluation as potential nonprotein calorie sources in patients with neurologic injury. Xylitol and fructose are not suitable since these substrates resulted in hyperglycemia and increased lactate accumulation in the central nervous system, and had detrimental effects on electrophysiologic recovery after ischemia. The short-chain fatty acids (acetate and butyrate) also had adverse effects on electrophysiologic recovery after ischemia, probably because of their hypotensive effects when given intravenously, rather than from the effects of their metabolism. (Crit Care Med 1994; 22:1841–1850)

The capacity of reducing-equivalent shuttles limits glycolysis during ethanol oxidation.

The inhibition of glycolysis during ethanol oxidation has been examined in isolated hepatocytes from fasted rats. Glycolytic flux was measured by determining the rate of release of tritium from [6-3H]glucose. During ethanol oxidation, the rate of glycolysis was inhibited 80% in freshly prepared hepatocytes, in which shuttle intermediates are depleted, but was depressed only about 20% in the presence of asparagine, a condition under which activity of the malate/aspartate shuttle was restored to normal levels. The inhibition of glycolysis was also partially released by addition of pyruvate and when alcohol dehydrogenase activity was depressed by 4-methylpyrazole. Titrations with this inhibitor revealed inverse linear relationships between the rates of glycolysis and ethanol oxidation. For any given rate of ethanol oxidation, glycolytic flux was lowest and the [lactate]/[pyruvate] ratio highest in the presence of aminooxyacetate, an inhibitor of the malate/aspartate shuttle, whereas flux was highest and the ratio lowest in the presence of asparagine. During these titrations with 4-methylpyrazole the inhibition of ethanol oxidation and concomitant restoration of glycolysis were accompanied by a decline in the [lactate]/[pyruvate] ratio, a substantial fall in the rate of reducing-equivalent transfer from cytoplasm to mitochondria and an increase in lactate accumulation. These findings imply that the reducing equivalents generated during ethanol oxidation compete with those arising in glycolysis for transfer to the mitochondria. This competition leads to an inhibition of aerobic glycolysis, and at the same time contributes to a rise in cytoplasmic NADH and fall in NAD+ that results in depression of anaerobic glycolysis. Allosteric inhibition of 6-phosphofructo-1-kinase due to a decrease in the concentration of fructose 2,6-bisphosphate did not appear to play a primary role in the inhibition of glycolysis by ethanol. Ethanol oxidation had no effect on glucose phosphorylation as measured with [2-3H]glucose, but induced a substantial increase in cycling between glucose and glucose 6-phosphate.

Lactate accumulation in isolated hypoxic rat ventricular myocardium: effect of different modulators of the cyclic GMP system.

Different agents which are known to increase tissue levels of cyclic guanosine 3':5'-monophosphate (cGMP), were found to decrease the lactate accumulation induced by hypoxia in isolated, non-beating rat myocardium from the right ventricle. One microM sodium nitroprusside increased the intracellular cGMP content 4 times during hypoxic conditions, and after 5 min. of hypoxia the intracellular lactate accumulation decreased by about 20%. 0.1 microM atrial natriuretic peptide increased cGMP 10 times during hypoxic conditions and decreased the lactate accumulation by about 40%. The reduction in lactate accumulation was mimicked by 1 mM 8-Br-cGMP and by Zaprinast (10 microM), a selective inhibitor of cGMP phosphodiesterase, which reduced lactate accumulation by 60% and 45%, respectively. Glyceryl trinitrate (1 nM and 1 microM) caused a slight increase in lactate accumulation both during normooxic and hypoxic conditions, but had no effect on tissue levels of cGMP. In conclusion, the results indicate that cyclic GMP reduces lactate accumulation in hypoxic, non-beating rat heart ventricular muscle and suggests that atrial natriuretic peptide, which is released from atrial tissue, may have beneficial metabolic effects on the heart.

Experimental autoimmune encephalomyelitis: An anatomically-based explanation of clinical progression in rodents

Lactate accumalation was measured soon after decapitation in three adjacent lower spinal cord regions of rats with EAE. Results indicate that during EAE, and in correlation with the onset of clinical signal of both initial attack and shorth-term relapse, a differential focal increase in lactate accumulation occurs in rat spinal cord compared to Freund's Complete Adjuvant controls, with greater increase occurring in more caudal segments. A [14C]antipyrine method of estimating relative spinal cord blood flow failed to find evidence that the lactate accumulations were due to focal ischemia. Subsequent measurement of isotopic water and total protein increases in the same cord regions indicated that a slight but significant increase in vasogenic edema occurs in correlation with the increase in lactate accumulation and the onset of EAE clinical signs. The data are interpreted as lending support to a speculative theory of paralysis induced by edema during EAE, in which nerve root endoneurium is postulated as the functionally vulnerable site. More specifically, it is hypothesized that the ascending progression of clinical signs of EAE in rodents can be explained on an anatomica basis by progressive disturbance of the nodes of Ranvier in nerve root myelinated fibers.

Some Metabolic Effects of Phenformin in Rat Adipose Tissue

In rat adipose tissue, phenformin (5 × 10−4M) inhibited the oxidation of C1 and C6 of glucose to CO2, blocked the stimulatory effect of insulin (32 μU./ml.) on Ct oxidation, inhibited lipogenesis from C1 and C6 of glucose, and abolished the stimulatory effect of acetate on glucose oxidation and lipogenesis. These effects were accompanied by lactate accumulation. Such inhibitory effects suggest that phenformin interrupts the pentose shunt and lipogenesis, both of which are pyridine nucleotide-linked processes. Phenformin was shown, by inhibition of oxidation of pyruvate C1, to interfere with the sequence at the point of pyruvate decarboxylation, since acetate incorporation was not affected. Both pyruvate decarboxylation and oxidation of acetate in the tricarboxylic acid cycle were blocked, presumably by interference with electron transport. This effect of phenformin on pyruvate decarboxylation is sufficient to explain the observed reduction in pentose shunt activity and lipogenesis as well as the increased lactate accumulation.

AEROBIC GLYCOLYSIS OF X-IRRADIATED THYMOCYTES

The aerobic lactate production of rat thymocyte suspensions incubated at 37 °C for 2 hours was doubled following exposure to approximately 70 r X-radiation. Lower doses down to 18 r also produced a significant increase in aerobic lactate production. Increased lactate accumulation following exposure to 1000 r was observed after incubation for as little as 30–60 minutes, though the rate of accumulation increased still further between 2 and 4 hours incubation. A decrease in pH or temperature during incubation of irradiated thymocyte suspensions minimized the lactic acid accumulation. A comparison of the effects of X-irradiation in different media and of the effects of several metabolic inhibitors suggested that the increase in aerobic lactate production was a sensitive indicator of cell damage associated with the loss of intracellular potassium ions.Respiration and anaerobic glycolysis of the thymus cells were both much less sensitive than aerobic glycolysis to the effects of X-irradiation. The "anaerobic" lactate production of rat thymocyte suspensions in the presence of dinitrophenol was reduced by 50% after exposure to 5000–6000 r of X-radiation.

AEROBIC GLYCOLYSIS OF X-IRRADIATED THYMOCYTES

The aerobic lactate production of rat thymocyte suspensions incubated at 37 °C for 2 hours was doubled following exposure to approximately 70 r X-radiation. Lower doses down to 18 r also produced a significant increase in aerobic lactate production. Increased lactate accumulation following exposure to 1000 r was observed after incubation for as little as 30–60 minutes, though the rate of accumulation increased still further between 2 and 4 hours incubation. A decrease in pH or temperature during incubation of irradiated thymocyte suspensions minimized the lactic acid accumulation. A comparison of the effects of X-irradiation in different media and of the effects of several metabolic inhibitors suggested that the increase in aerobic lactate production was a sensitive indicator of cell damage associated with the loss of intracellular potassium ions.Respiration and anaerobic glycolysis of the thymus cells were both much less sensitive than aerobic glycolysis to the effects of X-irradiation. The "anaerobic" lactate production of rat thymocyte suspensions in the presence of dinitrophenol was reduced by 50% after exposure to 5000–6000 r of X-radiation.