Control analysis as a tool to understand the formation of the las operon in Lactococcus lactis

In Lactococcus lactis the enzymes phosphofructokinase (PFK), pyruvate kinase (PK) and lactate dehydrogenase (LDH) are uniquely encoded in the las operon. We used metabolic control analysis to study the role of this organization. Earlier studies have shown that, at wild-type levels, LDH has no control over glycolysis and growth rate, but high negative control over formate production (C(Jformate)LDH=-1.3). We found that PFK and PK exert no control over glycolysis and growth rate at wild-type enzyme levels but both enzymes exert strong positive control on the glycolytic flux at reduced activities. PK exerts high positive control over formate (C(Jformate)PK=0.9-1.1) and acetate production (C(Jacetate)PK=0.8-1.0), whereas PFK exerts no control over these fluxes at increased expression. Decreased expression of the entire las operon resulted in a strong decrease in the growth rate and glycolytic flux; at 53% expression of the las operon glycolytic flux was reduced to 44% and the flux control coefficient increased towards 3. Increased las expression resulted in a slight decrease in the glycolytic flux. At wild-type levels, control was close to zero on both glycolysis and the pyruvate branches. The sum of control coefficients for the three enzymes individually was comparable with the control coefficient found for the entire operon; the strong positive control exerted by PK almost cancels out the negative control exerted by LDH on formate production. Our analysis suggests that coregulation of PFK and PK provides a very efficient way to regulate glycolysis, and coregulating PK and LDH allows cells to maintain homolactic fermentation during glycolysis regulation.     

Potential role for pyruvate kinase M2 in the regulation of murine cardiac glycolytic flux during in vivo chronic hypoxia

Carbohydrate metabolism in heart failure shares similarities to that following hypoxic exposure, and is thought to maintain energy homoeostasis in the face of reduced O₂ availability. As part of these in vivo adaptations during sustained hypoxia, the heart up-regulates and maintains a high glycolytic flux, but the underlying mechanism is still elusive. We followed the cardiac glycolytic responses to a chronic hypoxic (CH) intervention using [5-³H]-glucose labelling in combination with detailed and extensive enzymatic and metabolomic approaches to provide evidence of the underlying mechanism that allows heart survivability. Following 3 weeks of in vivo hypoxia (11% oxygen), murine hearts were isolated and perfused in a retrograde mode with function measured via an intraventricular balloon and glycolytic flux quantified using [5-³H]-glucose labelling. At the end of perfusion, hearts were flash-frozen and central carbon intermediates determined via liquid chromatography tandem mass spectrometry (LC-MS/MS). The maximal activity of glycolytic enzymes considered rate-limiting was assessed enzymatically, and protein abundance was determined using Western blotting. Relative to normoxic hearts, CH increased ex vivo cardiac glycolytic flux 1.7-fold with no effect on cardiac function. CH up-regulated cardiac pyruvate kinase (PK) flux 3.1-fold and cardiac pyruvate kinase muscle isoenzyme M2 (PKM2) protein content 1.4-fold compared with normoxic hearts. CH also augmented cardiac pentose phosphate pathway (PPP) flux, reflected by higher ribose-5-phosphate (R5P) content. These findings support an increase in the covalent (protein expression) and allosteric (flux) control of PKM2 as being central to the sustained up-regulation of the glycolytic flux in the chronically hypoxic heart.     

Effects of increased heart work on glycolysis and adenine nucleotides in the perfused heart of normal and diabetic rats

1. In the isolated perfused rat heart, the contractile activity and the oxygen uptake were varied by altering the aortic perfusion pressure, or by the atrial perfusion technique (;working heart'). 2. The maximum increase in the contractile activity brought about an eightfold increase in the oxygen uptake. The rate of glycolytic flux rose, while tissue contents of hexose monophosphates, citrate, ATP and creatine phosphate decreased, and contents of ADP and AMP rose. 3. The changes in tissue contents of adenine nucleotides during increased heart work were time-dependent. The ATP content fell temporarily (30s and 2min) after the start of left-atrial perfusion; at 5 and 10min values were normal; and at 30 and 60min values were decreased. ADP and AMP values were increased in the first 15min, but were at control values 30 or 60min after the onset of increased heart work. 4. During increased heart work changes in the tissue contents of adenine nucleotide and of citrate appeared to play a role in altered regulation of glycolysis at the level of phosphofructokinase activity. 5. In recirculation experiments increased heart work for 30min was associated with increased entry of [(14)C]glucose (11.1mm) and glycogen into glycolysis and a comparable increase in formation of products of glycolysis (lactate, pyruvate and (14)CO(2)). There was no major accumulation of intermediates. Glycogen was not a major fuel for respiration. 6. Increased glycolytic flux in Langendorff perfused and working hearts was obtained by the addition of insulin to the perfusion medium. The concomitant increases in the tissue values of hexose phosphates and of citrate contrasted with the decreased values of hexose monophosphates and of citrate during increased glycolytic flux obtained by increased heart work. 7. Decreased glycolytic flux in Langendorff perfused hearts was obtained by using acute alloxan-diabetic and chronic streptozotocin-diabetic rats; in the latter condition there were decreased tissue contents of hexose phosphates and of citrate. There were similar findings when working hearts from streptozotocin-diabetic rats with insulin added to the medium were compared with normal hearts. 8. The effects of insulin addition or of the chronic diabetic state could be explained in terms of an action of insulin on glucose transport. Increased heart work also acted at this site, but in addition there was evidence for altered regulation of glycolysis mediated by changes in tissue contents of adenine nucleotides or of citrate.     

Regulatory steps of glycolysis during environmental anoxia and muscular work in the cockle,Cardium tuberculatum: control of low and high glycolytic flux

Summary Concentrations of glycolytic intermediates, end products of anaerobic metabolism and the adenylates have been determined in the foot muscle and in the whole soft body tissue of the cockle,Cardium tuberculatum, after anoxic incubation and after the performance of vigorous escape movements. Comparison of the mass action ratios (MAR) with the equilibrium constants (Keq) showed that the reactions catalyzed by glycogen phosphorylase, hexokinase, phosphofructokinase (PFK) and pyruvate kinase (PK) were displaced from equilibrium under all physiological situations investigated.Changes in the levels of the glycolytic intermediates showed that activation of phosphofructokinase is largely responsible for the 100-fold increase of glycolytic flux in the foot muscle during exercise.Analysis of the whole soft body tissue showed that PFK is also involved in reduction of the glycolytic flux during anoxia, but a more pronounced change in the MAR occurs for PK, indicating that PK is strongly inhibited under these conditions.Differences in the regulation of glycolysis in muscular and non-muscular tissues can be related to changes in metabolite levels and to tissue-specific forms of pyruvate kinase with different regulatory properties.     

5-3H]glucose overestimates glycolytic flux in isolated working rat heart: role of the pentose phosphate pathway

We set out to study the pentose phosphate pathway (PPP) in isolated rat hearts perfused with [5-3H]glucose and [1-14C]glucose or [6-14C]glucose (crossover study with 1- then 6- or 6- then 1-14C-labeled glucose). To model a physiological state, hearts were perfused under working conditions with Krebs-Henseleit buffer containing 5 mM glucose, 40 microU/ml insulin, 0.5 mM lactate, 0.05 mM pyruvate, and 0.4 mM oleate/3% albumin. The steady-state C1/C6 ratio (i.e., the ratio from [1-14C]glucose to [6-14C]glucose) of metabolites released by the heart, an index of oxidative PPP, was not different from 1 (1.06 +/- 0.19 for 14CO2, and 1.00 +/- 0.01 for [14C]lactate + [14C]pyruvate, mean +/- SE, n = 8). Hearts exhibited contractile, metabolic, and 14C-isotopic steady state for glucose oxidation (14CO2 production). Net glycolytic flux (net release of lactate + pyruvate) and efflux of [14C]lactate + [14C]pyruvate were the same and also exhibited steady state. In contrast, flux based on 3H2O production from [5-3H]glucose increased progressively, reaching 260% of the other measures of glycolysis after 30 min. The 3H/14C ratio of glycogen (relative to extracellular glucose) and sugar phosphates (representing the glycogen precursor pool of hexose phosphates) was not different from each other and was <1 (0.36 +/- 0.01 and 0.43 +/- 0.05 respectively, n = 8, P < 0.05 vs. 1). We conclude that both transaldolase and the L-type PPP permit hexose detritiation in the absence of net glycolytic flux by allowing interconversion of glycolytic hexose and triose phosphates. Thus apparent glycolytic flux obtained by 3H2O production from [5-3H]glucose overestimates the true glycolytic flux in rat heart.     

Glucose requirement for postischemic recovery of perfused working heart

The quantitative importance of glycolysis in cardiomyocyte reenergization and contractile recovery was examined in postischemic, preload-controlled, isolated working guinea pig hearts. A 25-min global but low-flow ischemia with concurrent norepinephrine infusion to exhaust cellular glycogen stores was followed by a 15-min reperfusion. With 5 mM pyruvate as sole reperfusion substrate, severe contractile failure developed despite normal sarcolemmal pyruvate transport rate and high intracellular pyruvate concentrations near 2 mM. Reperfusion dysfunction was characterized by a low cytosolic phosphorylation potential [( ATP]/[( ADP][Pi]) due to accumulations of inorganic phosphate (Pi) and lactate. In contrast, with 5 mM glucose plus pyruvate as substrates, but not with glucose as sole substrate, reperfusion phosphorylation potential and function recovered to near normal. During the critical ischemia-reperfusion transition at 30 s reperfusion the cytosolic creatine kinase appeared displaced from equilibrium, regardless of the substrate supply. When under these conditions glucose and pyruvate were coinfused, glycolytic flux was near maximum, the glyceraldehyde-3-phosphate dehydrogenase/3-phosphoglycerate kinase reaction was enhanced, accumulation of Pi was attenuated, ATP content was slightly increased, and adenosine release was low. Thus, glucose prevented deterioration of the phosphorylation potential to levels incompatible with reperfusion recovery. Immediate energetic support due to maximum glycolytic ATP production and enhancement of the glyceraldehyde-3-phosphate dehydrogenase/3-phosphoglycerate kinase reaction appeared to act in concert to prevent detrimental collapse of [ATP]/[( ADP][Pi]) during creatine kinase dysfunction in the ischemia-reperfusion transition. Dichloroacetate (2 mM) plus glucose stimulated glycolysis but failed fully to reenergize the reperfused heart; conversely, 10 mM 2-deoxyglucose plus pyruvate inhibited glycolysis and produced virtually instantaneous de-energization during reperfusion. The following conclusions were reached. (1) A functional glycolysis is required to prevent energetic and contractile collapse of the low-flow ischemic or reperfused heart (2). Glucose stabilization of energetics in pyruvate-perfused hearts is due in part to intensification of glyceraldehyde-3-phosphate dehydrogenase/3-phosphoglycerate kinase activity. (3) 2-Deoxyglucose depletes the glyceraldehyde-3-phosphate pool and effects intracellular phosphate fixation in the form of 2-deoxyglucose 6-phosphate, but the cytosolic phosphorylation potential is not increased and reperfusion failure occurs instantly. (4) Consistent correlations exist between cytosolic ATP phosphorylation potential and reperfusion contractile function. The findings depict glycolysis as a highly adaptive emergency mechanism which can prevent deleterious myocyte deenergization during forced ischemia-reperfusion transitions in presence of excess oxidative substrate.     

Creatine kinase increases the solubility and enzymatic activity of pyruvate kinase by means of diazymatic coupling

Five separate methods, paper chromatography, ethanol solubility, pH dependence, enzymatic activity, and NMR saturation transfer, were used to study the coupling of pyruvate kinase (PK) and creatine kinase (CK). Each method demonstrated specific coupling of the two proteins. The coupling with CK showed that PK had increased ethanol solubility, enzymatic activity, pH-dependent aqueous solubility, and ability to receive ATP directly from CK even at very high (1.2 M) ionic strength at equilibrium conditions. The robustness of the coupling between two enzymes with highin vivo concentrations indicates the necessity for considering kinetic schemes utilizing high enzyme concentrations, as opposed to the dilute properties of Michaelis — Menten kinetics. The direct transfer of ATP from PK to CK constitutes a net transfer of the glycolytically produced high-energy phosphate onto creatine, maintaining a high ADP and low ATP concentration in the vicinity of the glycolytic complex, conditions suited to maintaining a high glycolytic flux. The demonstration of the physical and functional coupling of two soluble enzymes, termed diazymes, suggests that glycolysis may be regulated by diazymatic coupling and that other cellular processes could be regulated similarly.     

Early pre-diabetic state alters adaptation of myocardial glucose metabolism during ischemia in rats

Pre-diabetic subjects with high insulin secretory capacity have double risk of cardiovascular disease compared with subjects who do not develop insulin-resistance. It is well established that the ability of the myocardium to increase its glycolytic ATP production plays a crucial role in determining cell survival under conditions of ischemia. Up to now, whether the pre-diabetic state reduces the tolerance of the heart to ischemia by affecting its ability to increase its energy production through glycolysis remains unknown. The aim of the present study was to assess whether insulin resistance affects the ability of the myocardium to increase glycolysis under ischemic conditions. Male Wistar rats were fed for 8 weeks a fructose-enriched (33%) diet to induce a pre-diabetic state. Hearts were isolated and subjected to ex-vivo low-flow (2%) ischemia for 30 min. The fructose diet increased sarcolemmal GLUT4 localisation in myocardial cells under basal conditions compared with controls. This effect was not accompanied by increased glucose utilisation. Ischemia induced the translocation of GLUT4 to the plasma membrane in controls but did not significantly modify the distribution of these transporters in pre-diabetic hearts. Glycolytic flux under ischemic conditions was significantly lower in fructose-fed rat hearts compared with controls. The reduction of glycolytic flux during ischemia in fructose-fed rat hearts was not due to metabolic inhibition downstream hexokinase II since no cardiac accumulation of glucose-6-phosphate was detected. In conclusion, our results suggest that the pre-diabetic state reduces the tolerance of the myocardium to ischemia by decreasing glycolytic flux adaptation.     

A modelling study of feedforward activation in human erythrocyte glycolysis

Though feedforward activation (FA) is a little known principle of control in metabolic networks, there is one well-known example; namely, the activation of pyruvate kinase (PK) by fructose-1,6-biphosphate (FBP) in glycolysis. The effects of this activation on the enzyme's kinetics are well characterised, but its possible role in glycolytic control has not been determined, and, experimentally, there is as yet no direct way of modifying the enzyme to remove just the FBP activation without affecting other aspects of the enzyme's kinetics. Given this limitation, we used a detailed numerical simulation of human erythrocyte glycolysis to simulate the effects of selective removal of the activation of PK by FBP on steady-state metabolite concentrations and on the dynamic response of glycolytic flux to a sudden increase of the cell's demand for ATP. Our modelling results predict that in the absence of FA steady-state levels of metabolites within the activation loop, i.e. from FBP to phosphoenolpyruvate, would be four- to thirteen-fold higher than normal, whereas levels of ATP and metabolites outside the loop, i.e. glucose-6-phosphate, fructose-6-phosphate and pyruvate, would be lower than normal. Existing clinical evidence in a patient with haemolytic anaemia, correlated with a lack of activation of PK by FBP (Paglia D.E., Valentine W.N., Holbrook C.T., Brockway R., Blood (1983) 62 972-979), is consistent with this prediction. In response to changing demand for ATP, the model predicts that the corresponding change of glycolytic flux would entail changes of metabolite concentrations in the absence of FA, but that in its presence the levels of metabolites within the activation loop remain essentially unperturbed. Thus, our results suggest that by stabilising metabolite pools in the face of variable glycolytic flux, FA may serve to avoid perturbations of the oxygen affinity of haemoglobin (sensitive to the levels of 2,3-phosphoglycerate) and of cell osmolality that would otherwise occur during variations in the cell's demand for ATP. In addition, by significantly raising the steady-state setpoint of intermediate metabolite pools, the productivity index (ratio of glycolytic flux to total metabolites in the pathway) of glycolysis would fall almost four-fold in the absence of forward activation.     

Pyrophosphate and fructose 2,6-bisphosphate effects on glycolysis in pea seed extracts

The participation of pyrophosphate-dependent phosphofructokinase (PPi-PFK) in plant glycolysis was examined using extracts from pea seeds (Pisum sativum L. cv Alaska). Glycolysis starting with fructose 6-phosphate was measured under aerobic conditions as the accumulation of pyruvate. Pyruvate accumulated in a medium containing PPi and adenosine diphosphate at about two-thirds of the rate in a medium containing adenosine diphosphate and adenosine triphosphate (ATP). The PPi-dependent pyruvate accumulation had the same reactant requirements and sensitivity to glycolysis inhibitors, sodium fluoride, and iodoacetamide, as the well-established ATP-dependent glycolysis. Added fructose 2,6-bisphosphate stimulated both the PPi-dependent pyruvate accumulation and PPi-PFK activity whereas this modulator had no effect on ATP-dependent glycolysis or ATP-PFK. Collectively these results demonstrate a PPi-dependent glycolytic pathway in plants which is responsive to fructose 2,6-bisphosphate.     

Mechanisms of glycolytic control during hibernation in the ground squirrel Spermophilus lateralis

Summary The mechanisms of glycolytic rate control during hibernation in the ground squirrel Spermophilus lateralis were investigated in four tissues: heart, liver, kidney, and leg muscle. Overall glycogen phosphorylase activity decreased significantly in liver and kidney to give 50% or 75% of the activity found in the corresponding euthermic organs, respectively. The concentration of fructose-2,6-bisphosphate (F-2,6-P₂) decreased significantly in heart and leg muscle during hibernation to 50% and 80% of euthermic tissue concentrations, respectively, but remained constant in liver and kidney. The overall activity of pyruvate dehydrogenase (PDH) in heart and kidney from hibernators was only 4% of the corresponding euthermic values. Measurements of phosphofructokinase (PFK) and pyruvate kinase (PK) kinetic parameters in euthermic and hibernating animals showed that heart and skeletal muscle had typical rabbit skeletal M-type PFK and M₁-type PK. Liver and kidney PFK were similar to the L-type enzyme from rabbit liver, whereas liver and kidney PK were similar to the M₂ isozyme found primarily in rabbit kidney. The kinetic parameters of PFK and PK from euthermic vs hibernating animals were not statistically different. These data indicate that tissue-specific phosphorylation of glycogen phosphorylase and PDH, as well as changes in the concentration of F-2,6-P₂ may be part of a general mechanism to coordinate glycolytic rate reduction in hibernating S. lateralis.Abbreviations ADP adenosine diphosphate - AMP adenosine monophosphate - ATP adenonine triphoshate - EDTA ethylenediaminetetra-acetic acid - EGTA ethylene glycol tetra-acetic acid - F-6-P fructose 6-phosphate - F-1,6-P₂ fructose 1,6-bisphosphate - F-2,6-P₂ fructose-2,6-bisphosphate - K _a activation coefficient - I₅₀ concentration of inhibitor which reduces control activity by 50% - PDH pyruvate dehydrogenase - PEP phosphoenolpyruvate - PFK 6-phosphofructo-1-kinase - PK pyruvate kinase     

Nandrolone-pretreatment enhances cardiac beta(2)-adrenoceptor expression and reverses heart contractile down-regulation in the post-stress period of acute-stressed rats

To investigate whether nandrolone decanoate (ND)-pretreatment can modulate (1) beta-adrenoceptor expression and (2) myocardial contractility in response to beta-adrenoceptors stimulation with isoproterenol (ISO), in hearts of both normal and stressed rats. Rats were treated with 15 mg/(kgday) of Deca-Durabolin (ND, 1 ml i.m.) or with vehicle (oil) for 14 days. The day after the last injection, the dose-response to ISO (1 x 10(-8), 5 x 10(-8) and 10(-7)M), was studied in isolated rat hearts harvested from unstressed animals (unstressed+vehicle (control) or unstressed+ND) or from stressed animals (stressed+vehicle or stressed+ND): acute stress protocol consisted in restrain for 1h immediately before sacrifice. ND-pretreatment increased beta(2)-adrenoceptor expression. In baseline conditions all hearts had a similar left ventricular developed pressure (LVDP) and maximum rate of increase of LVDP (dP/dt(max)). In hearts of unstressed+vehicle or unstressed+ND, ISO caused a similar increase in LVDP (+90-100%) and dP/dt(max) (+120-150%). However, hearts of stressed+vehicle animals showed a marked depression of inotropic response to ISO (i.e. for ISO 1 x 10(-8),-55% in LVDP response versus unstressed). Yet, in hearts of stressed+ND-animals the effect of stress was reversed, showing the highest response to ISO (i.e. for ISO 1 x 10(-7), +30% LVDP response versus unstressed). The ND-induced beta(2)-adrenoceptor overexpression does not affect ISO-response in unstressed animals. However, acute stress induces a down-regulation of ISO-response, which is reversed by ND-pretreatment. Since the physiological post-stress down-regulation of adrenergic-response is absent after nandrolone treatment, the heart may be exposed to a sympathetic over-stimulation. This might represent a risk for cardiovascular incidents in anabolic steroid addicts under stressing conditions.     

Na+/H+ exchange inhibition with cariporide prevents alterations of coronary endothelial function in streptozotocin-induced diabetes

Hyperglycemia encountered during diabetes triggers abnormalities of vascular function associated with cell acidosis and calcium overload. The purpose of this study was to determine, whether Na⁺/H⁺ exchanger (NHE-1) inhibition by cariporide protects coronary cells against the deleterious effect of hyperglycemia in the rat. In vivo hyperglycemia was triggered by streptozotocin injection. One week after, the glycemia was checked and the control and diabetic animals were treated or not with cariporide (2.5 mg/kg/day) for two weeks. Glycemia was again estimated and the hearts were perfused according to the Langendorff mode at forced flow. The left ventricle developed pressure (LVDP) and heart rate (HR) were determined with a latex balloon inserted into the left ventricle. Coronary pressure was artificially increased to 130 mmHg by infusing a thromboxane A₂ analogue (U46619). This allowed the evaluation of endothelium-dependent (EDD) and endothelium-independent (EID) dilatation through bolus injections of carbamoylcholin and sodium nitroprusside, respectively. Releases of lactate and pyruvate in the coronary effluents were also determined. Diabetes did not modified LVDP, but reduced HR (−15%). This was associated with a marked decrease in EDD (−56%) and EID (−30%), while the cytosolic redox potential (estimated as the lactate/pyruvate ratio) was reduced. NHE-1 inhibition restored EDD and the lactate/pyruvate ratio without improving EID and HR. The present findings indicate that NHE-1 exchanger inhibition by cariporide protects the coronary endothelium against the deleterious effects of hyperglycemia.     

Contribution of adenosine to changes in coronary flow in metabolically stimulated rat heart

The extent to which endogenous, extracellular adenosine mediates increased coronary flow in crystalloid-perfused, isovolumic rat hearts stimulated with either norepinephrine or isoproterenol was examined. When infused into the coronary circulation, norepinephrine (1 x 10(-7) M) rapidly increased left ventricular developed pressure (LVDP) from 81 +/- 6 to 235 +/- 13 mmHg (1 mmHg = 133.3 Pa) and coronary flow from 12.7 +/- 0.8 to 18.4 +/- 0.7 mL.min-1.g-1. The presence of either adenosine deaminase (2 U.mL-1) or the adenosine receptor antagonist, 8-phenyltheophylline (5 x 10(-6) M) in the perfusate of norepinephrine-stimulated hearts augmented the increase in LVDP and +/- dP/dtmax by 10-20% but reduced the increase in coronary flow by 34%. Doubling the rate of adenosine deaminase infusion, or infusing the enzyme and 8-phenyltheophylline together did not alter their inhibitory effectiveness. Similar results were observed with hearts stimulated with isoproterenol (5 x 10(-8) M). These data show that about a third of the vasodilation that results from the metabolic stimulation of rat heart by catecholamines is due to the receptor-mediated action of extracellular adenosine.     

Studies on the pigeon red blood cell metabolism

1. Pigeon erythrocyte was found to depend on the glycolytic and pentose phosphate pathway for most of its energy production in the form of adenosine triphosphate and reducing potential, since there was no detectable activity of any of the citric acid cycle (TCA) cycle enzymes measured. 2. The absence of detectable amounts of 2,3-diphosphoglyceric acid (2-3-DPG) indicated that there is no direct relationship between the active glycolytic system and the function of these cells. 3. A comparison of the mass action ratios with the equilibrium constants of the glycolytic reactions showed that hexokinase, phosphofructokinase and pyruvate kinase reactions are displaced from equilibrium, implying that these are the key regulatory enzymes of glycolysis in pigeon erythrocytes. 4. The changes in the concentrations of the glycolytic metabolites under hypoxic conditions that stimulate the flux through the glycolytic pathway were found to be consistent with the above hypothesis. 5. Flux measurements of the pentose phosphate pathway showed that it metabolizes only 3.4% of the total glucose consumed by the resting erythrocyte. 6. Hypoxic conditions resulted in a stimulation of the pentose phosphate pathway by as much as four-fold, whilst the glycolytic pathway was not stimulated by more than about twice.     

Activation of glycolysis by insulin with a sequential increase of the 6-phosphofructo-2-kinase activity, fructose-2,6-bisphosphate level and pyruvate kinase activity in cultured rat hepatocytes

The involvement of 6-phosphofructo-2-kinase, fructose 2,6-bisphosphate [Fru(2,6)P2] and pyruvate kinase in the insulin-dependent short-term activation of glycolysis was studied in primary cultures of rat hepatocytes. The short-term influence of insulin on these parameters was dependent on the insulin concentration used for the long-term culture. Cells were cultured either with 10 nM or 0.1 nM insulin for 48 h, and are referred to as 'insulin cells' and 'control cells', respectively. Insulin cells exhibited a high level of Fru(2,6)P2. Addition of insulin to insulin cells led to an immediate stimulation of glycolysis (two-fold) and activation of pyruvate kinase. The concentration of Fru(2,6)P2 and activity of 6-phosphofructo-2-kinase remained constant. Control cells exhibited a very low level of Fru(2,6)P2 and low activity of 6-phosphofructo-2-kinase directly after the medium change. However, both parameters increased during a 1-2-h incubation in the absence of insulin. Although the level of Fru(2,6)P2 thus changed up to tenfold the glycolytic rate remained at a constant value. Addition of insulin to control cells led to a 5-8-fold stimulation of glycolysis but only after a 30-90-min lag phase. During this lag period insulin strongly increased sequentially the 6-phosphofructo-2-kinase, the level of Fru(2,6)P2 and the pyruvate kinase activity. The activation of the latter enzyme slightly preceded the onset of the insulin-stimulated glycolysis. Addition of insulin to control cells, which were preincubated for 3 h in the absence of insulin and in which the Fru(2,6)P2 level had risen insulin-independently, led to an immediate increase in glycolysis without a lag phase. It is concluded that in this insulin-sensitive cell system: the changes of glycolytic flux did not correlate with changes in the level of total Fru(2,6)P2 either in insulin or in control cells; an increase in the Fru(2,6)P2 concentration was not obligatory for the insulin-dependent stimulation of glycolysis in insulin cells; activation of pyruvate kinase and thus glycolysis by insulin did not proceed unless the Fru(2,6)P2 level had been elevated above a threshold level. The lack of correlation between total Fru(2,6)P2 levels and the glycolytic flux and the apparent existence of a threshold concentration for Fru(2,6)P2 suggest a permissive action for this effector in enzyme interconversion.     

Substrate-induced alterations of high energy phosphate metabolism and contractile function in the perfused heart

The bioenergetic basis by which the Krebs cycle substrate pyruvate increased cardiac contractile function over that observed with the Embden-Meyerhof substrate glucose was investigated in the isovolumic guinea pig heart. Alterations in the content of the high energy phosphate metabolites and the rate of high energy phosphate turnover were measured by 31P NMR. These were correlated to the changes in contractile function and rates of myocardial oxygen consumption. Maximum left ventricular developed pressure (LVDP) and high energy phosphates were observed with 16 mM glucose or 10 mM pyruvate. In hearts perfused with 16 mM glucose, the intracellular phosphocreatine (PCr) concentration was 15.2 +/- 0.6 mM with a PCr/Pi ratio of 10.3 +/- 0.9. The O2 consumption was 5.35 mumol/g wet weight/min, and these hearts exhibited a LVDP of 97 +/- 3.7 mm Hg at a constant paced rate of 200 beats/min. In contrast, when hearts were switched to 10 mM pyruvate, the PCr concentration was 18.3 +/- 0.4 mM, the PCr/Pi ratio was 30.4 +/- 2.2, the O2 consumption was 6.67 mumol/g wet weight/min, and the LDVP increased to 125 +/- 3.3 mm Hg. From NMR saturation transfer experiments, the steady-state flux of ATP synthesis from PCr was 4.9 mumol/s/g of cell water during glucose perfusion and 6.67 mumol/s/g of cell water during pyruvate perfusion. The flux of ATP synthesis from ADP was measured to be 0.99 mumol/s/g of cell water with glucose and calculated to be 1.33 mumol/s/g of cell water with pyruvate. These results suggest that pyruvate quite favorably alters myocardial metabolism in concert with the increased contractile performance. Thus, as a mechanism to augment myocardial performance, pyruvate appears to be unique.     

Endothelial NO formation does not control myocardial O2 consumption in mouse heart

To test whether endothelium-derived nitric oxide (NO) regulates mitochondrial respiration, NO was pharmacologically modulated in isolated mouse hearts, which were perfused at constant flow to sensitively detect small changes in myocardial O2 consumption (MVO2). Stimulation of NO formation by 10 microM bradykinin (BK) increased coronary venous nitrite release fivefold to 58 +/- 33 nM (n = 17). Vasodilatation by BK, adenosine (1 microM), or papaverine (10 microM) decreased perfusion pressure, left ventricular developed pressure (LVDP), and MVO2. In the presence of adenosine-induced vasodilatation, stimulation of endothelial NO synthesis by BK had no effect on LVDP and MVO2. Also, inhibition of NO formation by NG-monomethyl-l-arginine (l-NMMA, 100 microM) did not significantly alter LVDP and MVO2. Similarly, intracoronary infusion of authentic NO 2 microM were contractile dysfunction and MVO2 reduction observed. Because BK-induced stimulation of endothelial NO formation and basal NO are not sufficient to impair MVO2 in the saline-perfused mouse heart, a tonic control of the respiratory chain by endothelial NO is difficult to conceive.     

Substrate-dependent functional defects and altered mitochondrial respiratory capacity in hearts from guinea pigs with iron deficiency anemia

Iron deficiency anemia was induced by dietary means in weanling guinea pigs. A 25% higher ventricular wall mass per 100 g body mass was seen after 6 weeks of feeding. Myocardial performance was determined in isolated perfused hearts using an isovolumic Langendorff preparation. All hearts exhibited a 25% decrease in left ventricular developed pressure (LVDP) and decreased dP/dt when substrate was switched from 10 mM pyruvate to 16.6 mM glucose. The glucose reduction in LVDP resulted from decreased systolic pressure, which completely reversed when hearts again metabolized pyruvate. With glucose as substrate, left ventricular developed pressure-end diastolic volume relationships were indistinguishable. However, with pyruvate, iron-deficient hearts appeared to be less responsive to the increased energy demands required by elevated diastolic volumes. Rates of state 3 respiration were 18% below control with glutamate + malate as substrate, and 38% lower with pyruvate + malate in mitochondria isolated from anemic animals. No differences in respiration were noted with succinate. Cytochrome a + a3 content, cytochrome oxidase activity and total mitochondrial protein content appeared to be unchanged. In contrast, cytochromes b, c + c1, and the flavoproteins were significantly decreased. The data suggest that iron deficiency anemia induces cardiac hypertrophy with a fixed but defective mitochondrial population, potentially placing the heart in an energetic imbalance. These differences in mitochondrial function were expressed by decreased myocardial performance when the heart metabolizes pyruvate, an exclusively aerobic substrate.