Pyruvate reverses fatty-acid-induced depression of ventricular function and calcium overload after hypothermia in guinea pig hearts

OBJECTIVE: High levels of free fatty acids have been shown to impair mechanical recovery and calcium homeostasis of isolated rat hearts following hypothermic perfusion. The objective of the present study was to investigate whether inhibition of fatty acid oxidation through activation of pyruvate dehydrogenase by millimolar concentrations of pyruvate could influence functional recovery and Ca2+ homeostasis after a hypothermic insult. METHODS: Ventricular function and myocardial calcium ([Ca]total) were measured in 3 different groups of Langendorff-perfused guinea pig hearts exposed to 40 min hypothermic (15 degrees C) perfusion, followed by 30 min rewarming at 37 degrees C. The hearts were perfused with either 11.1 mM glucose (G), glucose and 1.2 mM palmitate (GP), or glucose, palmitate and 5 mM pyruvate (GPP) as energy substrates. RESULTS: All groups showed marked elevations in [Ca]total during hypothermia (from 0.6-0.7 mumol.g dry wt-1 to 9.3-12.2 mumol.g dry wt-1 at 40 min hypothermia, P < 0.05), associated with a pronounced increase in left ventricular end-diastolic pressure (LVEDP from 0-2 to 50-60 mmHg). Following rewarming, GP-perfused hearts showed significantly lower recovery of mechanical function compared to both G- and GPP-perfused hearts (% recovery of left ventricular developed pressure: 27 +/- 8 vs. 62 +/- 3 and 62 +/- 8%, respectively, P < 0.05). The reduced mechanical recovery of GP-perfused hearts was associated with elevated [Ca]total. In separate experiments we found that addition of 1.2 mM palmitate reduced glucose oxidation ([14C]glucose) from 1.77 +/- 0.28 mumol.min-1.g dry wt-1 (G-perfused hearts) to 0.15 +/- 0.04 mumol.min-1.g dry wt-1 (GP-perfused hearts, P < 0.05), implying that fatty acids had become the major substrate for oxidative phosphorylation. Fatty acid oxidation was, however, less pronounced after further addition of 5 mM pyruvate. Thus, palmitate oxidation ([3H]palmitate) was more than 40% lower in GPP-perfused than in GP-perfused hearts (0.83 +/- 0.22 vs. 1.41 +/- 0.12 mumol.min-1.g dry wt-1, P < 0.05). CONCLUSIONS: The present results demonstrate impaired ventricular function and calcium homeostasis after hypothermia in guinea pig hearts perfused with fatty acids in addition to glucose, as compared to hearts perfused with glucose alone. Furthermore, we show that these unfavourable effects of fatty acids can be overcome by an exogenous supply of pyruvate.     

Evaluation of the protective efficacy of a recombinant dengue envelope B domain fusion protein against dengue 2 virus infection in mice

1. The influence of Ca2+ on the effects of glucagon on glycolysis was investigated in the isolated perfused rat liver. Livers from fed rats were perfused in an open system with Krebs/Henseleit-bicarbonate buffer (pH 7.4). Glucose release, lactate plus pyruvate production (glycolysis) and oxygen uptake were measured. The following results were obtained: 2. In livers perfused with Ca(2+)-free Krebs/Henseleit-bicarbonate buffer and after depletion of the intracellular pools, the initial and transient stimulation of glycolysis, which is normally observed shortly after the onset of glucagon infusion, was more pronounced when compared to livers perfused with normal perfusion fluid (2.5 mM Ca2+) and without previous depletion of the intracellular pools (controls); the subsequent inhibition of glycolysis was delayed in Ca(2+)-free perfused livers and was less pronounced in comparison with the controls at the end of the glucagon infusion period (20 min). 3. Perfusion with a Ca(2+)-free medium supplemented with EDTA, without previous depletion of the intracellular pools, also produced a substantial reduction in the effects of glucagon on glycolysis. 4. Ca(2+)-free perfusion did not affect the stimulative action of glucagon on glucose release (glycogenolysis) and oxygen uptake. 5. Glycolysis inhibition by cAMP also was abolished in Ca(2+)-free perfused livers, and the initial stimulation was enhanced. 6. Mn2+, a metal ion known as a competitor of Ca2+, considerably reduced the action of glucagon on glycolysis; Mn2+ did not affect the basal rates of glycolysis. 7. Sr2+, a metal ion that is often recognized as Ca2+ by several biological structures and processes, increased the inhibitory action of glucagon on glycolysis. 8. Several organic compounds, which directly or indirectly take part in Ca2+ fluxes, were also able to diminish (e.g., verapamil) or even to abolish (carbenoxolone) the inhibitory action of glucagon on glycolysis. 9. It was concluded that, under the conditions of the living cell, Ca2+ is important for glycolysis inhibition by glucagon. In principle at least, the results can be explained in terms of the known Ca2+ dependencies of several protein kinases and protein phosphatases.     

Comparative effects of englitazone and glyburide on gluconeogenesis and glycolysis in the isolated perfused rat liver

Englitazone (CP 68,722, Pfizer) is a member of a family of drugs known as thiazolidinediones. One member of this family, troglitazone (Rezulin), is currently utilized in the treatment of Type 2 diabetes. Previous studies have focused on the ability of englitazone to increase insulin sensitivity in various tissues. However, little information is available regarding the direct effect of englitazone on hepatic glucose metabolism in the absence of insulin. Therefore, the following studies were conducted to comparatively evaluate the effect of englitazone and glyburide (a representative sulfonylurea) on gluconeogenesis and glycolysis from various substrates in the isolated perfused rat liver (IPRL). In isolated perfused rat livers of 24-hr fasted rats infused with lactate (2 mM), englitazone (6.25 to 50 microM) produced a concentration-dependent decrease (32-93%) in hepatic gluconeogenesis. When dihydroxyacetone (1 mM) and fructose (1 mM) were used as metabolic substrates, englitazone inhibited gluconeogenesis by 31 and 15%, respectively, while increasing glycolysis by 42 and 50%. Similar effects on gluconeogenesis and glycolysis were observed with glyburide, even though the effects with glyburide were more acutely evident, reversible, and of a greater magnitude. Such data suggest alterations in hepatic glucose production may contribute to the decrease in plasma glucose concentrations observed in individuals treated with englitazone and glyburide. These alterations may include effects on several regulatory enzymes (e.g. fructose-1,6-bisphosphatase, pyruvate kinase, and phosphoenolpyruvate carboxykinase), which warrant further investigation.     

Altered hypoxia-induced coronary vasodilatation in diabetic rabbit heart

The effect of type 1 diabetes mellitus on hypoxia-induced coronary vasodilation was studied in isolated perfused rabbit hearts. Four groups of hearts were compared: control hearts from normal rabbits perfused with physiological buffer (5 mM glucose and 2 mM pyruvate added), hearts from alloxan-induced diabetic rabbits (same perfusion as control), hyperglycemic hearts from normal rabbits perfused with 22 mM glucose and 2 mM pyruvate, and hyperosmotic hearts from normal rabbits perfused with 5 mM glucose, 2 mM pyruvate, and 8.5 mM choline chloride. Hypoxia was produced by perfusion with a mixture of N2- and O2- saturated solutions. Endothelium-dependent and -independent dilators were also tested. Papaverine-induced coronary vasodilatation was unaltered, whereas that of serotonin and adenosine was significantly reduced in hyperglycemic and hyperosmotic hearts but not in diabetic hearts perfused with normoglycemic buffer. Hypoxia (PO2 from 515 +/- 86 to 131 +/- 24 mmHg; 1 mmHg = 133.3 Pa) caused a significant coronary vasodilatation in normal hearts (-66 +/- 3%). This vasodilatation was reduced slightly in diabetic (-45 +/- 7%, p < 0.05) and severely in hyperglycemic (-21 +/- 5%, p < 0.05) and hyperosmotic (-24 +/- 5%, p < 0.05) hearts. The adenosine-receptor antagonist 8-phenyltheophylline (10 microM) reduced hypoxia-induced vasodilatation in normal and diabetic hearts. However, inhibition of prostaglandin synthesis with diclofenac (1 microM), which reduces hypoxia-induced vasodilatation in normal hearts, had no effect in diabetic hearts. In conclusion, alloxan-induced type 1 diabetes mellitus in rabbits is accompanied by a reduced coronary vasodilator response to hypoxia. The contribution of adenosine in this response is unaffected. However, the abated contribution of cyclooxygenase products may account for the reduced vasodilatation during hypoxia in this particular model.     

Control of glucose utilization in working perfused rat heart

Metabolic control analyses of glucose utilization were performed for four groups of working rat hearts perfused with Krebs-Henseleit buffer containing 10 mM glucose only, or with the addition of 4 mM D-beta-hydroxybutyrate/1 mM acetoacetate, 100 nM insulin (0.05 unit/ml), or both. Net glycogen breakdown occurred in the glucose group only and was converted to net glycogen synthesis in the presence of all additions. The flux of [2-3H]glucose through P-glucoisomerase (EC 5.3.1.9) was reduced with ketones, elevated with insulin, and unchanged with the combination. Net glycolytic flux was reduced in the presence of ketones and the combination. The flux control coefficients were determined for the portion of the pathway involving glucose transport to the branches of glycogen synthesis and glycolysis. Major control was divided between the glucose transporter and hexokinase (EC 2.7.1.1) in the glucose group. The distribution of the control was slightly shifted to hexokinase with ketones, and control at the glucose transport step was abolished in the presence of insulin. Analysis of the pathway from 3-P-glycerate to pyruvate determined that the major control was shared by enolase (EC 4.2.1.1) and pyruvate kinase (EC 2.7.1.40) in the glucose group. Addition of ketones, insulin, or the combination shifted the control to P-glycerate mutase (EC 5.4.2.1) and pyruvate kinase. These results illustrate that the control of the metabolic flux in glucose metabolism of rat heart is not exerted by a single enzyme but variably distributed among enzymes depending upon substrate availability, hormonal stimulation, or other changes of conditions.     

Regulation of exogenous and endogenous glucose metabolism by insulin and acetoacetate in the isolated working rat heart. A three tracer study of glycolysis, glycogen metabolism, and glucose oxidation

Myocardial glucose use is regulated by competing substrates and hormonal influences. However, the interactions of these effectors on the metabolism of exogenous glucose and glucose derived from endogenous glycogen are not completely understood. In order to determine changes in exogenous glucose uptake, glucose oxidation, and glycogen enrichment, hearts were perfused with glucose (5 mM) either alone, or glucose plus insulin (40 microU/ml), glucose plus acetoacetate (5 mM), or glucose plus insulin and acetoacetate, using a three tracer (3H, 14C, and 13C) technique. Insulin-stimulated glucose uptake and lactate production in the absence of acetoacetate, while acetoacetate inhibited the uptake of glucose and the oxidation of both exogenous glucose and endogenous carbohydrate. Depending on the metabolic conditions, the contribution of glycogen to carbohydrate metabolism varied from 20-60%. The addition of acetoacetate or insulin increased the incorporation of exogenous glucose into glycogen twofold, and the combination of the two had additive effects on the incorporation of glucose into glycogen. In contrast, the glycogen content was similar for the three groups. The increased incorporation of glucose in glycogen without a significant change in the glycogen content in hearts perfused with glucose, acetoacetate, and insulin suggests increased glycogen turnover. We conclude that insulin and acetoacetate regulate the incorporation of glucose into glycogen as well as the relative contributions of exogenous glucose and endogenous carbohydrate to myocardial energy metabolism by different mechanisms.     

The effect of 2,4-dinitrophenol and glucose on the utilization of succinate, lactate, pyruvate and formate by Escherichia coli K12

The effect of protonophore 2,4-dinitrophenol (5-25 microM) in lithium chloride acidic medium (pH 4.5) on utilization of acidic energetic substrates was studied in the presence and absence of glucose. The rate of succinate, lactate and pyruvate utilization by AB 1157 strains was two times lower, than by the KS 400 strain. Isomolar glucose concentration (0.5 mM) decreased the utilization rate of succinate by 60% pyruvate by 30% the AB 1157 strain being much more sensitive than the KS 400 strain both to the action of glucose and to the reaction to glucose and 2,4-dinitrophenol. Acidification of the medium by bacteria has been observed, which shows changes in energy metabolism (glycolysis takes place instead of oxidation of substrates) under conditions of intensive aeration. The results obtain permit assuming that the AB 1157 strain has a certain defect of the membrane which causes increased proton conduction.     

Heat stress induces rapid recovery of mechanical function of ischemic fatty acid perfused hearts by stimulating glucose oxidation during reperfusion

Whole-body heat stress (HS) in rats leads to the accumulation of myocardial heat shock proteins and subsequent protection against ischemic injury in glucose-perfused hearts. We determined whether HS treatment would confer protection against ischemia in hearts perfused with high levels of fatty acids. In addition, since fatty acids can potentiate ischemic injury by inhibiting glucose metabolism, the effects of HS on glucose utilization were also determined. Anesthetized rats were subjected to whole-body hyperthermia by raising body temperature to 41-42 degrees C 15 min. Twenty four hours later, their hearts were perfused with buffer containing either 11 mM glucose alone or 11 mM glucose and 1.2 mM palmitate, and then subjected to ischemic conditions followed by reperfusion. In hearts perfused with glucose only, HS improved aortic flow (expressed as percent change from preischemic aortic flow) late into the reperfusion period. Rates of overall glucose utilization under these conditions were similar between control and HS hearts. When hearts were perfused with 1.2 mM palmitate, the benefits of HS on aortic flow occurred at the onset of the reperfusion period. This beneficial effect was associated with a significant increase in glucose oxidation. Our results show that HS induces a faster rate of recovery in fatty acid perfused hearts but does not offer more protection against ischemic damage when compared with hearts perfused with glucose as a sole substrate.     

A 13C mass isotopomer study of anaplerotic pyruvate carboxylation in perfused rat hearts

Anaplerotic pyruvate carboxylation was examined in hearts perfused with physiological concentrations of glucose, [U-13C3]lactate, and [U-13C3]pyruvate. Also, a fatty acid, [1-13C]octanoate, or ketone bodies were added at concentrations providing acetyl-CoA at a rate resulting in either low or substantial pyruvate decarboxylation. Relative contributions of pyruvate and fatty acids to citrate synthesis were determined from the 13C labeling pattern of effluent citrate by gas chromatography-mass spectrometry (see companion article, Comte, B., Vincent, G., Bouchard, B., and Des Rosiers, C. (1997) J. Biol. Chem. 272, 26117-26124). Precision on flux measurements of anaplerotic pyruvate carboxylation depended on the mix of substrates supplied to the heart. Anaplerotic fluxes were precisely determined under conditions where acetyl-CoA was predominantly supplied by beta-oxidation, as it occurred with 0.2 or 1 mM octanoate. Then, anaplerotic pyruvate carboxylation provided 3-8% of the OAA moiety of citrate and was modulated by concentrations of lactate and pyruvate in the physiological range. Also, the contribution of pyruvate to citrate formation through carboxylation was equal to or greater than through decarboxylation. Furthermore, 13C labeling data on tissue citric acid cycle intermediates and pyruvate suggest that (i) anaplerosis occurs also at succinate and (ii) cataplerotic malate decarboxylation is low. Rather, the presence of citrate in the effluent perfusate of hearts perfused with physiological concentrations of glucose, lactate, and pyruvate and concentrations of octanoate leading to maximal oxidative rates suggests a cataplerotic citrate efflux from mitochondria to cytosol. Taken altogether, our data raise the possibility of a link between pyruvate carboxylation and mitochondrial citrate efflux. In view of the proposed feedback regulation of glycolysis by cytosolic citrate, such a link would support a role of anaplerosis and cataplerosis in metabolic signal transmission between mitochondria and cytosol in the normoxic heart.     

Intracellular redox state and control of gluconeogenesis in perfused chicken liver

The role of the cellular redox state in the control of gluconeogenesis was studied in hemoglobin-free perfused chicken liver, by fluorimetric measurement of the redox states of intracellular pyridine nucleotides. The aminotransferase inhibitor, aminooxyacetate, completely inhibited gluconeogenesis from lactate in the perfused rat liver and to a small extent in the perfused chicken liver. In chicken liver, the highest rate of glucose production was seen with lactate, followed by fructose, pyruvate, and glycerol. When compared at 5 mM, the rate of glucose production from pyruvate was only 10% of that from lactate. Glucose production from a pyruvate/lactate mixture decreased with increasing proportions of pyruvate, together with redox changes of pyridine nucleotides to a more oxidized state. Increased reduction of pyridine nucleotides upon infusion of ethanol was associated with an increased glucose production from pyruvate, and the increase was abolished during octanoate infusion. This abolishment was accompanied by an increase in the acetoacetate to beta-hydroxybutyrate ratio with an oxidation of pyridine nucleotides. The octanoate-inhibited gluconeogenesis occurred at the higher lactate concentration (10 mM) with a transient oxidation of pyridine nucleotides. No significant inhibition was observed at 1 mM lactate, although an instant reduction of pyridine nucleotides was taking place. The rate of beta-hydroxybutyrate generation during octanoate infusion was 2.2 times higher at 1 mM than at 10 mM lactate. The inhibitory effect of octanoate on glyconeogenesis was completely relieved by the addition of NH4Cl. The results demonstrate that the regeneration of NADH in the cytosol is limited in chicken liver, and that gluconeogenesis is regulated, in part, by alteration in the redox states of mitochondria and cytosol.     

Estimates of glycolysis, pyruvate (de)carboxylation, pentose phosphate pathway, and methyl succinate metabolism in incapacitated pancreatic islets

Pancreatic islets were cultured for 24 h in the presence of 1 mM glucose, which renders islets incapable of responding to glucose with insulin release. These islets were compared to islets maintained at 20 mM glucose for 24 h. Detritiation of [2-3H]glucose and [5-3H]glucose in 1 mM glucose islets was normal, suggesting that glucose transport and phosphorylation and all enzymes of glycolysis were not down-regulated in the incapacitated islets. 14CO2 formation from [U-14C]glucose and [6-14C]glucose was inhibited up to 80% and 14CO2 from methyl succinate was inhibited up to 60%, indicating that down-regulation at (a) mitochondrial site(s) might explain the incapacitated insulin release. 14CO2 formation from [3,4-14C]glucose (which becomes [1-14C]pyruvate) was decreased, indicating that the reaction catalyzed by pyruvate dehydrogenase was down-regulated. This decrease, however, was not as large as the decreases in 14CO2 formation from [U-14C]glucose, [2-14C]glucose (which becomes [2-14C]pyruvate), or [6-14C]glucose (which becomes [3-14C]pyruvate), indicating that other reactions were also down-regulated. 14CO2 formation from [1-14C]glucose was inhibited less than that from [6-14C]glucose in the incapacitated islets (34 vs 54%) and these rates indicated that flux of glucose through the pentose phosphate pathway was increased in the incapacitated islet, such that 29% (0.4 nmol of 1.4 glucose/100 islets/90 min) was metabolized via this pathway in the incapacitated islet but only 3.4% (0.1 of 2.9 nmol glucose/100 islets/90 min) was metabolized via the pentose pathway in the 20 mM glucose islets. With rates of 14CO2 evolved from glucose labeled at C2 and C6 and from methyl succinate labeled at C1 + C4 and C2 + C3 the 14CO2 ratio formula was used to calculate the ratios of carboxylated and decarboxylated pyruvate. Roughly equal amounts of pyruvate entered the citric acid cycle by each route in islets maintained for 24 h at 1, 5, or 20 mM glucose. The results indicate that decarboxylation and carboxylation of pyruvate were about equally suppressed in incapacitated islets and that direct inhibition of reactions of the cycle was unlikely. This is consistent with evidence which indicates that down-regulation of both pyruvate carboxylase and pyruvate dehydrogenase occurs in incapacitated islets, i.e., under long-term conditions that modify amounts of enzymes (MacDonald et al., 1991, J. Biol. Chem. 266, 22392-22397).(ABSTRACT TRUNCATED AT 400 WORDS)     

Regulation of energy metabolism of the heart during acute increase in heart work

We determined the contribution of all major energy substrates (glucose, glycogen, lactate, oleate, and triglycerides) during an acute increase in heart work (1 microM epinephrine, afterload increased by 40%) and the involvement of key regulatory enzymes, using isolated working rat hearts exhibiting physiologic values for contractile performance and oxygen consumption. We accounted for oxygen consumption quantitatively from the rates of substrate oxidation, measured on a minute-to-minute basis. Total beta-oxidation (but not exogenous oleate oxidation) was increased by the work jump, consistent with a decrease in the level of malonyl-CoA. Glycogen and lactate were important buffers for carbon substrate when heart work was acutely increased. Three mechanisms contributed to high respiration from glycogen: 1) carbohydrate oxidation was increased selectively; 2) stimulation of glucose oxidation was delayed at glucose uptake; and 3) glycogen-derived pyruvate behaved differently from pyruvate derived from extracellular glucose. Despite delayed activation of pyruvate dehydrogenase relative to phosphorylase, glycogen-derived pyruvate was more tightly coupled to oxidation. Also, glycogen-derived lactate plus pyruvate contributed to an increase in the relative efflux of lactate versus pyruvate, thereby regulating the redox. Glycogen synthesis resulted from activation of glycogen synthase late in the protocol but was timed to minimize futile cycling, since phosphorylase a became inhibited by high intracellular glucose.     

Competition between lactate and fatty acids as sources of ATP in the isolated working rat heart

Fatty acid oxidation is generally considered the major source of energy in the heart, although lactate oxidation can be a major contributor to ATP production, depending on the concentration and availability of other competing substrates. In this study, isolated working rat hearts were used to directly determine the relationship between lactate and fatty acid oxidation to overall ATP production from exogenous sources. A range of lactate from 0.5 to 8.0 mM lactate was added to hearts perfused with buffer containing 5.5 mM glucose, and either 0.4 or 1.2 mM palmitate over a 100 min period. Rates of glycolysis, glucose oxidation, lactate oxidation, and palmitate oxidation were determined. In the presence of 0.5 mM lactate and 0.4 mM palmitate, lactate oxidation provided 17% of the ATP production and palmitate oxidation provided 68%, with the remainder coming from glucose oxidation and glycolysis. In the presence of 0.4 mM palmitate, an increase in lactate from 0.5 to 8.0 mM increased the steady state rates of lactate oxidation from 1239+/-236 to 5247+/-940 nmol/min/g dry weight, respectively. The contribution of lactate oxidation to total ATP production increased to 37%, with palmitate oxidation now contributing only 52% of the total ATP produced. At 8.0 mM lactate and 1.2 mM palmitate, lactate oxidation contributed 13% of the total ATP production, while palmitate oxidation contributed 81%. This data demonstrates that under near physiological conditions of lactate (0.5 mM) and fatty acids (0.4 mM), the preferred energy substrate of the heart remains to be fatty acids, and that only at high levels of lactate, such as can be observed during exercise or severe stress, does lactate oxidation become a significant source of ATP production.     

Ser644 is important for catalytic activity but is not involved in cAMP-dependent phosphorylation of yeast 6-phosphofructo-2-kinase

To identify the target amino acid for the cAMP-dependent phosphorylation of yeast 6-phosphofructo-2-kinase Ser644 was mutated to Ala. The plasmid-encoded wild-type and mutant enzymes were overexpressed in E. coli TG2 cells and in the yeast strain DFY658. Like the wild-type enzyme, the Ser644-->Ala mutant was phosphorylated in vivo after addition of glucose to yeast cells and in vitro by the catalytic subunit of protein kinase A. The specific activity of the mutant enzyme was 6-fold lower than that of the wild-type yeast 6-phosphofructo-2-kinase, but both enzymes were activated in response to the addition of glucose to yeast cells.     

Peculiarities of the action of protein positively reacting in the sedimentation test for cancer on the activity of glycolytic enzymes

Blood serum of oncologic patients due to immunoglobulin involved in its composition, activates glycolysis in the soluble fraction of muscles when using starch, glycogen and glucose as substrates. The activation is registered under both aerobic and anaerobic conditions. When elucidating the immunoglobulin effect in a glycolytic chain under aerobic conditions it is shown that its activating effect in the incomplete incubation system is manifested with such glycolysis substrates as fructose-6-phosphate and 2-phosphoglyceric acid. Glycolysis activation with serum is insignificant or absent at all with the presence of glucose-6-phosphate, fructose-1,6-diphosphate, 3-phosphoglyceric aldehide, 3-phosphoglyceric acid, phosphoenolpyruvic acid, sodium pyruvate. Immunoglobulin isolated from the blood serum of oncologic patients does not affect the activity of purified preparations of hexokinase, glycerinaldehydephosphate dehydrogenase, lactate dehydrogenase under aerobic and anaerobic conditions. When using the air as a gas medium lactate dehydrogenase is activated by immunoglobulin. Lactate dehydrogenase activity under aerobic and anaerobic conditions is essentially lower than in the case when the air serves as a gas medium.     

Myocardial glucose uptake and breakdown during adenosine-induced vasodilation

In isolated K+ (16.2 mM)-arrested cat hearts perfused at constant pressure adenosine infusions (0.8 mumoles - min-1 - 100 g-1 for 10 min) caused an increase in myocardial 14C-glucose uptake and release of 14CO2 + H14CO3- AND 14C-lactate simultaneously with a rise in coronary flow. The ratio of the release of 14CO2 + H14CO3- to that of 14C-lactate and the specific activity of lactate in the effuate were not altered. In K+ -arrested hearts perfused with constant volume neither glucose uptake nor glucose breakdown were influenced by 0.8 or 100 mumoles - min-1 - 100 g-1 adenosine with 0.1 - 5 mM glucose in the perfusion medium. It is concluded that adenosine does not affect directly the myocardial glucose carrier system, aerobic or anaerobic glucose breakdown or glycogenolysis, but enhances glucose uptake secondarily by increasing coronary flow. This interpretation is substantiated by the finding that mechanically produced increases in perfusion volume caused similar increases in myocardial glucose uptake as were observed with comparable adenosine-induced coronary flow increments.     

Performance and cost-effectiveness of selective screening criteria for Chlamydia trachomatis infection in women. Implications for a national Chlamydia control strategy

At 9 mM glucose, experimental results show that mitochondrial phosphate depletion (induced by glucose phosphorylation, catalyzed by mitochondrial hexokinase) reduces the activities of the respiratory chain, oxidative phosphorylation, and glutaminase. Consequently, the 14C-lactate oxidation to 14CO2 is lowered in the presence of glucose. The fall of ATP level triggers a high aerobic glycolysis by deinhibiting fructose-6-P kinase. NADH, generated by enhanced glyceraldehyde-3-P dehydrogenase activity, increases the reducing power. Moreover, the lactate dehydrogenase (LDH) system is shifted toward lactate formation, while NAD+ is regenerated and the oligomycin-inhibited ATP production is replaced by the iodoacetate-inhibited ATP production. From 14CO2 production and lactate accumulation it is calculated that about 60% of 14C-glucose which disappears is channelled into extraglycolytic reactions. On the contrary, 82% of glucose below l mM is metabolized through non-glycolytic reactions. The pyruvate kinase-M2 (PK-M2) inhibition does not limit the glycolytic flow from 9 mM glucose, but it may cause sustained gluconeogenesis.     

Suppression of insulin-stimulated phosphatidylinositol 3-kinase activity by the beta3-adrenoceptor agonist CL316243 in rat adipocytes

Insulin increased 2-deoxyglucose (2-DG) uptake via the translocation of glucose transporter (GLUT) 4 to the plasma membrane fraction in rat adipocytes. The stimulatory actions of insulin were accompanied by both an increase in the immunoreactive p85 subunit of phosphatidylinositol (PI) 3-kinase in the plasma membrane fractions and PI 3-kinase activation by tyrosine phosphorylation of the p85 subunit. The beta3-adrenoceptor agonist CL316243 (CL) suppressed all the insulin actions in adenosine deaminase (ADA)-treated cells, but was without effect in non-ADA-treated cells. The inhibitory effects of CL on GLUT 4 translocation and PI 3-kinase activation were abolished by the addition of N6-phenylisopropyl adenosine. Cholera toxin treatment, which markedly increased intracellular cAMP levels, suppressed increases in the levels of GLUT 4 and PI 3-kinase in the plasma membrane fractions in response to insulin. In addition, dibutyryl (Bt2) cAMP also impaired the activation of PI 3-kinase by insulin. These results indicated that CL suppressed insulin-stimulated glucose transport under conditions where cAMP levels were markedly increased (approximately 12-fold). The inhibitory actions of PI 3-kinase activation by insulin were exerted even when cAMP, 8-bromo-cAMP, or Bt2 cAMP was added to immunoprecipitates of the p85 subunit of PI 3-kinase, after treating the cells with insulin. These results suggest that CL suppressed insulin-stimulated PI 3-kinase activity via a cAMP-dependent mechanism, at least in part, direct cAMP action in ADA-treated adipocytes, by which PI 3-kinase activation was inhibited, resulting in the decrease in GLUT 4 translocation and subsequent 2-DG uptake in response to insulin.     

Stimulation of weak organic acid uptake in rat renal tubules by cadmium and nystatin

The uphill uptake of a weak organic acid, fluorescein, in superficial proximal tubules of the rat kidney was stimulated by CdCl2 (0.1 mM) or nystatin (20 microM) in the absence of metabolic substrates in the incubation medium. The stimulation could be observed during the initial period of incubation (up to 30 min) only and was prevented completely by ouabain (0.1 mM), fluoroacetate (1 mM), malonate (10 mM), alpha-cyano-4-hydroxycinnamate (0.1 mM), phenylpyruvate (1 mM), D-malate (2 mM) or phenazine methosulfate (20 microM). In the renal cortex fragment suspension, both Cd2+ and nystatin increased the ouabain-sensitive, basal oxygen consumption and inhibited the rate of glucose production from pyruvate, but not from lactate. In the presence of lactate (0.5-5 mM) in the incubation medium, Cd2+ and nystatin rather inhibited fluorescein uptake, while externally added pyruvate did not influence their stimulatory effects. Taken together, these data suggest that both activation of the tricarboxylic acid cycle and export of reducing equivalents from the mitochondria to the cytosol are necessary for the stimulatory effects of Cd2+ and nystatin on the weak organic acid uptake to develop.