Glycolytic Substrates Research Articles

High-fat feeding inhibits exercise-induced increase in mitochondrial respiratory flux in skeletal muscle

Twenty one healthy untrained male subjects were randomized to follow a high-fat diet (HFD; 55-60E% fat, 25-30E% carbohydrate, and 15E% protein) or a normal diet (ND; 25-35E% fat, 55-60E% carbohydrate, and 10-15E% protein) for 2(1/2) wk. Diets were isocaloric and tailored individually to match energy expenditure. At 2(1/2) wk of diet, one 60-min bout of bicycle exercise (70% of maximal oxygen uptake) was performed. Muscle biopsies were obtained before and after the diet, immediately after exercise, and after 3-h recovery. Insulin sensitivity (hyperinsulinemic-euglycemic clamp) and intramyocellular triacylglycerol content did not change with the intervention in either group. Indexes of mitochondrial density were similar across the groups and intervention. Mitochondrial respiratory rates, measured in permeabilized muscle fibers, showed a 31 ± 11 and 26 ± 9% exercise-induced increase (P < 0.05) in state 3 (glycolytic substrates) and uncoupled respiration, respectively. However, in HFD this increase was abolished. At recovery, no change from resting respiration was seen in either group. With a lipid substrate (octanoyl-carnitine with or without ADP), similar exercise-induced increases (31-62%) were seen in HFD and ND, but only in HFD was an elevated (P < 0.05) respiratory rate seen at recovery. With HFD complex I and IV protein expression decreased (P < 0.05 and P = 0.06, respectively). A fat-rich diet induces marked changes in the mitochondrial electron transport system protein content and in exercise-induced mitochondrial substrate oxidation rates, with the effects being present hours after the exercise. The effect of HFD is present even without effects on insulin sensitivity and intramyocellular lipid accumulation. An isocaloric high-fat diet does not cause insulin resistance.

A NOVEL TIME-SHARED FLUOROMETER GIVES THE MITOCHONDRIAL REDOX STATE AS THE RATIO OF TWO COMPONENTS OF THE RESPIRATORY CHAIN OF THE ANIMAL AND HUMAN BUCCAL CAVITY WITH QUANTITATIVE MEASURES OF THE REDOX ENERGY STATE

The source of energy for life is the tissue mitochondria and they demand a complex chain of biochemicals to ensure proper physiological function. Classically, the blood levels, and not the tissue levels of these metabolites, are determined by expensive and time-consuming biochemical analyses. Since the tissue mitochondria are the consumers of the substrates of glycolysis and of fatty acid metabolism, their redox state is a unique accessible monitor of tissue metabolism and its blockade due to toxins.

Silencing of glycolysis in muscle: experimental observation and numerical analysis

The longstanding problem of rapid inactivation of the glycolytic pathway in skeletal muscle after contraction was investigated using (31)P NMR spectroscopy and computational modelling. Accumulation of phosphorylated glycolytic intermediates (hexose monophosphates) during cyclic contraction and subsequent turnover during metabolic recovery was measured in vivo in human quadriceps muscle using dynamic (31)P NMR spectroscopy. The concentration of hexose monophosphates in muscle peaked 40 s into metabolic recovery from maximal contractile work at 6.9 +/- 1.3 mm (mean +/- s.d.; n = 8) and subsequently declined at a rate of 0.009 +/- 0.001 mm s(1). It was next tested whether the current knowledge of the kinetic controls in the glycolytic pathway in muscle integrated in the Lambeth and Kushmerick computational model of skeletal muscle glycolysis explained the experimental data. It was found that the model underestimated the magnitude of deactivation of the glycolytic pathway in resting muscle, resulting in depletion of glycolytic intermediates and substrate for oxidative ATP synthesis. Numerical analysis of the model identified phosphofructokinase and pyruvate kinase as the kinetic control sites involved in deactivation of the glycolytic pathway. Ancillary 100-fold inhibition of both phosphofructokinase and pyruvate kinase was found necessary to predict glycolytic intermediate and ADP concentrations correctly in resting human muscle. Incorporation of this information into the model resulted in highly improved agreement between predicted and measured in vivo dynamics of hexose monophosphates in muscle following contraction. We concluded that silencing of the glycolytic pathway in muscle following contraction is most likely to be mediated by phosphofructokinase and pyruvate kinase inactivation on a time scale of seconds and minutes, respectively, and is necessary to prevent depletion of vital cellular substrates.

Cord Blood Units Collected with Liquid CPD Appear to Contain Significantly More Nucleated and CD34+ Cells Than Units Collected with Dry Heparin.

Abstract 4227Transplantation of Cord Blood Stem Cells depends primarily on the number and potency of stem cells harvested, processed and cryopreserved. Previous studies have not compared the anti-coagulant used in collection systems with respect to their impact on the cord blood unit cell count and composition. We compared cord blood units collected in Citric Phosphate with Dextrose Buffer (CPD) or Heparin by measuring the pre-processed total nucleated cell count (TNC), post-processed TNC, % CD34 and total number of CD34+ cell numbers from units harvested randomly in remote hospitals in the US. Between August 2005 and May 2009, cord blood units harvested in collection bags containing lyophilized (dry) Heparin (N=65) and cord blood units harvested in FDA approved Sentinel collection bags containing 35 ml of CPD (N>6300) were processed using standard processing methods which included either RBC depletion or volume reduction based on initial collected volume. We observed significantly greater pre-processed TNC count (9.59 +/- 5.98 × 108 Vs 7.36 +/- 4.96 × 108, p=0.003), post-processing TNC count (7.72 +/- 4.61 × 108 Vs 5.80 +/- 3.63 × 108, p=0.001), % CD34+ (0.46 +/- 0.28 Vs 0.37 +/- 0.23, p=0.024) and number of CD34+ cells (2.72 +/- 2.73 × 106 Vs 1.72 +/- 1.44 × 106, p=0.003) in the CPD units. Interestingly, viability by dye exclusion was 7% higher (p=0.0001) in the post-processed Heparin units despite more pre-TNC (by 24%), post-TNCs (by 29%), and more %CD34+ (by 22%) and CD34+ cells (by 54%) in the CPD units. Post-processed CD34+ measurements relied upon gating to select live cells (by 7AAD exclusion) and reflect a decrease in viable CD34+ cells in Heparin units. Together, these results indicate that units collected using CPD contain significantly more TNCs prior to and after processing than units collected using Heparin. Furthermore, both the % CD34+ and total number of CD34+ cells were significantly lower in the Heparin containing units. Both TNC and CD34+ values are primary indicators of graft potency in clinical studies. Cord blood units collected in the pubic cord blood system and the vast majority of cord blood units used in clinical transplantation have been collected in CPD. CPD is both an anti-coagulant and a preservative as it contains Dextrose which provides a substrate for glycolysis and preserves the metabolism in cells. Heparin, which does not contain Dextrose to preserve cell metabolism, is rarely used as an anti-coagulant for blood collection, as it can only be useful for blood that is to be transfused within 12 hours of collection because it is broken down over longer periods of time. Also, the use of dry Heparin may adversely affect the osmolarity of the cord blood unit, thereby compromising cellularity. However, further studies are recommended to elucidate the mechanism by which dry Heparin may adversely affect the TNC and CD34 content of cord blood units. Disclosures:Kraus:PerkinElmer/Viacord: Employment. Foster:PerkinElmer/Viacord: Employment. Bridges:PerkinElmer/Viacord: Employment. Walters:PerkinElmer/Viacord: Membership on an entity's Board of Directors or advisory committees.

The Auxiliary Substrate Concept: From simple considerations to heuristically valuable knowledge

AbstractMicroorganisms are used in biotechnology. They are either (i) aim and purpose of a process, e.g. with the production of single cell proteins, or (ii) mean to an end insofar as they serve as a catalyst or “factory” for syntheses (e.g. of products of primary and secondary metabolism, of enzymes and antibiotics) or for the degradation and detoxification of harmful organics and inorganics. In all cases, the efficiency and velocity, finally the productivity, are parameters which essentially determine the economy of the processes. Therefore, search for approaches to optimize these processes is a permanent task and challenge for scientists and engineers. It is shown that the auxiliary substrate concept is suitable to increase the yield coefficients. It is based on the energetic evaluation of organics, on the knowledge that organics as sources of carbon and energy for growth are deficient in ATP and/or reducing equivalents, and says that it is possible to improve the carbon conversion efficiency up to the carbon metabolism determined upper limit. The latter is determined by inevitable losses of carbon along the way of assimilation and anabolism and amounts to about 85% for so‐called glycolytic substrates, e.g. glucose, methanol, and to about 75% for gluconeogenetic substrates, e.g. C2‐substrates (acetic acid, hexadecane). The approach is explained and some experimental examples are presented. By simultaneous utilization of an extra energy source (auxiliary substrate) the yield coefficient can be increased (i) in glucose from about 0.5 to 0.7 g/g (by means of formate), (ii) in acetate from 0.34–0.4 to 0.5–0.65 g/g (by means of formate and thiosulfate, respectively), and (iii) in hexadecane from about 0.94 to 1.26 g/g (by means of formate). The precalculated yield coefficients and mixing ratios agree well with the experimentally attained ones. The approach is easily feasible and economically valuable.

Glycolysis and Respiration in Mouse Ldhc -/- Sperm.

Targeted mutation of the lactate dehydrogenase type C gene (Ldhc) reduced considerably glucose consumption in sperm, resulting in decreased ATP production and loss of sperm motility. Upon backcrossing the mutation onto different genetic backgrounds for ten generations (KOF10), we observed significant fertility differences between C57Bl/6N (KOF10/C57) and 129SvEv (KOF10/129) strain males. While KOF10/C57 males were subfertile, KOF10/129 males were infertile. Sperm analyses revealed that KOF10/129 sperm had significantly lower ATP levels and progressive motility than KOF10/C57 sperm. Inhibition of glycolysis with sodium iodoacetate further lowered ATP levels and motility in KOF10/C57, but not in KOF10/129 sperm. These results suggest that KOF10/C57 sperm continue to produce significantly more ATP by glycolysis than KOF10/129 sperm, presumably explaining why a few pups were sired by KOF10/C57 but not by KO10/129 male mice. This difference between KOF10/C57 and KOF10/129 might be explained by a metabolic difference found by NMR spectroscopy between 129SvEv and C57Bl/6N WT sperm: glucose utilization was approximately 4 times lower in 129SvEv WT sperm (3.84±1.15 nmol glucose consumed/hour/106 spermatozoa) compared to C57Bl/6N WT sperm (12.93±3.48). However, there were no significant differences in fertility of WT 129SvEv and C57Bl/6N males, indicating that while glycolysis is essential for sperm function, a higher rate does not necessarily result in higher sperm fertilizing ability. A positive correlation has been reported between sperm quality and the efficiency of respiration, and this implies that sperm rely on respiration to support high levels of ATP production. Because of the extremely low level of ATP produced by glycolysis in KOF10/129 sperm, they provide a good model for examining the role of respiration in sperm motility. Sperm from WT 129SvEv and KOF10/129 mice were incubated with lactate or pyruvate as mitochondrial substrates, glucose as a glycolytic substrate, or a mix of lactate plus glucose. High levels of ATP and increases in progressive motility were observed in KOF10/129 and WT sperm incubated with mitochondrial substrates, and these effects were abrogated by the mitochondrial respiratory uncoupler CCCP. As expected, ATP levels and motility were extremely low when KOF10/129 sperm were incubated with glucose. However, the presence of lactate, as a mitochondrial substrate in medium containing glucose, did not rescue the KOF10/129 phenotype: respiration as measured by ATP production was inactive in the presence of glucose. The respiration rate was determined by measuring oxygen (O2) consumption, and was high when WT or KOF10/129 sperm were incubated with lactate alone, and lowered by alpha-cyano-4-hydroxycinnamic acid (inhibitor of mitochondrial lactate and pyruvate transport). However glucose or deoxy-glucose in combination with lactate caused a reduction in O2 consumption (putative Crabtree effect). These results strongly suggest that in mouse sperm the presence of glucose induces a shift to the glycolytic pathway. This also occurs when glycolysis is inhibited in KOF10/129 sperm. Further studies on the mechanism of this regulation and its physiological role are in progress. (poster)

Investigations on the motility of human spermatozoa in a defined medium in the presence of metabolic inhibitors and of carnitine.

When washed human sperm were incubated in a modified Krebs-Ringer buffer solution in the absence of exogenous metabolizable substrates at 30 degrees C they maintained progressive motility for at least six hours. Under these conditions the spermatozoa apparently utilize endogenous substrates but addition of exogenous substrates (glucose, fructose, acetate, short-chain fatty acids, branched chain amino acids) did not affect the % progressive motility or % total motility of the cells. The phospholipase inhibitors, quinacrine and Upjohn No. 1002, inhibited progressive motility when added to sperm utilizing endogenous substrate, and subsequent addition of oxidative or glycolytic substrates did not reverse the inhibition. In contrast, the inhibition by KCN of progressive motility based upon utilization of endogenous substrate was reversed upon addition of glycolyzable compounds (glucose or fructose). The addition of carnitine or its acetyl-, propionyl-, isobutyryl-, valeryl- or isovaleryl esters did not consistently affect progressive or total motility of sperm samples. The inhibitor, octylsulfobetaine, inhibited sperm motility at a concentration higher than that required for inhibition of carnitine acetyltransferase or translocase activity. On the basis of these results it does not appear that exogenous carnitine has an effect on the motility of human sperm incubated under the conditions described here.

Inhibition of human muscle-specific enolase by methylglyoxal and irreversible formation of advanced glycation end products

Methylglyoxal (MG) was studied as an inhibitor and effective glycating factor of human muscle-specific enolase. The inhibition was carried out by the use of a preincubation procedure in the absence of substrate. Experiments were performed in anionic and cationic buffers and showed that inhibition of enolase by methylglyoxal and formation of enolase-derived glycation products arose more effectively in slight alkaline conditions and in the presence of inorganic phosphate. Incubation of 15 micromolar solutions of the enzyme with 2 mM, 3.1 mM and 4.34 mM MG in 100 mM phosphate buffer pH 7.4 for 3 h caused the loss a 32%, 55% and 82% of initial specific activity, respectively. The effect of MG on catalytic properties of enolase was investigated. The enzyme changed the KM value for glycolytic substrate 2-phospho-D-glycerate (2-PGA) from 0.2 mM for native enzyme to 0.66 mM in the presence of MG. The affinity of enolase for gluconeogenic substrate phosphoenolpyruvate altered after preincubation with MG in the same manner, but less intensively. MG has no effect on Vmax and optimal pH values. Incubation of enolase with MG for 0-48 h generated high molecular weight protein derivatives. Advanced glycation end products (AGEs) were resistant to proteolytic degradation by trypsin. Magnesium ions enhanced the enzyme inactivation by MG and facilitated AGEs formation. However, the protection for this inhibition in the presence of 2-PGA as glycolytic substrate was observed and AGEs were less effectively formed under these conditions.

AMP-Dependent Protein Kinase Activators

See related articles, pages 403–411 A patent inability to properly use glycolytic and fatty acid substrates and progressive insulin resistance characterize non–insulin dependent diabetes mellitus. Curiously, the failing myocardium may also share these unfavorable characteristics. Insights from the clinical arena indicate a significant relationship between insulin resistance and New York Heart Association heart failure classification.1 In fact, insulin resistance may beget heart failure and heart failure may beget insulin resistance.2 Fueled by a recent resurgence in studies of the metabolic derangements contributing to cardiovascular disease, we are beginning to comprehend the complexities of metabolism or at least appreciate the bounds of our ignorance. Our understanding of such issues surrounding diabetes and heart failure is predicated on identifying the proximal regulators of substrate availability, sensing, and utilization. One potentially satisfying candidate appears to be AMP-dependent protein kinase (AMPK)3,4 and is the focus of the study by Gundewar et al in this issue of Circulation Research .5 AMPK has emerged as a principal figure in the story of metabolic regulation and dysregulation in diabetes, exercise, and, to a lesser extent, myocardial ischemia. AMPK activation appears to be beneficial in the context of myocardial ischemia and reperfusion, at least in terms of infarct size reduction. Such findings are confirmed by the present5 and previous studies.6 However, the intriguing element of the present study5 is the significant effect of chronic, postischemic treatment with the AMPK-activating biguanide metformin in the context of the failing heart (Figure). Despite a prior contraindication for heart failure because of concerns about the generation of lactic acidosis, interest has been renewed (although it actually never waned for some determined investigators) for the potential use of the insulin “sensitizer” metformin in diabetics with heart failure. Such interest is based not simply on the …

Evidence of Cardiomyocyte Fructose Uptake as an Alternate Glycolytic Substrate

Evidence of Cardiomyocyte Fructose Uptake as an Alternate Glycolytic Substrate

203 EFFECT OF FRUCTOSE ON THE DEVELOPMENT OF BOVINE SEXED EMBRYOS IN VITRO

It is important to optimize the condition of culture to promote the development of embryos fertilized by sex-sorted sperm. Fructose is a monosaccharide hexose that can be used as a substrate for glycolysis. Recently, there has been interest in exploring the embryotrophic properties of fructose during bovine embryo culture. The present study was undertaken to ascertain the effect of supplementation of fructose in culture on the development of sexed embryos in vitro. A total of 2310 slaughterhouse dairy oocytes, spread over 3 experimental replicates, were subjected to routine in vitro maturation (IVM) for 22 h. The maturation medium was M-199 with Earl’s salts, L-glutamine, 26.19 mm sodium bicarbonate, and 25 mm HEPES plus 10% fetal bovine serum. Fertilization of IVM oocytes was performed in Brackett and Oliphant’s medium by using frozen and sorted X-sperm from a single bull. Groups of 50 matured oocytes were added to a final volume of 100 μL of fertilization medium, with a final concentration of 0.3 × 106 spermatozoa mL–1. The oocytes were incubated with X-sperm for 6 h in 5% CO2 humidified air at 39°C. Presumptive zygotes were randomly allocated to culture either in a control medium (6 mg mL–1 of BSA CR1aa medium) or with 4.5 mm fructose added to CR1aa culture medium for 7 days in an atmosphere of 5% O2, 5% CO2, and 90% N2 at 39°C. Embryo cleavage, and development to the morulae and blastocyst stages were evaluated on Days 2, 5, and 7 of culture, respectively. Embryos were graded according to International Embryo Transfer Society standards. The total number of blastocysts recorded was the sum of grade C1 and C2 embryos. Data were analyzed by using the General Linear Model (SPSS 11.0, SPSS Inc., Chicago, IL, USA). As shown in Table 1, addition of fructose to the culture medium affected neither the rate of cleavage nor subsequent development to the morula stage or total blastocyst development. However, the proportion of C1-grade embryos of the total blastocysts was significantly (P &lt; 0.05) improved. This study suggests that addition of fructose to the culture medium can promote embryo quality and yields that are suitable for embryo transfer. Table 1.Effect of addition of fructose on in vitro development of bovine sexed embryos

The effect of UCP3 overexpression on mitochondrial ROS production in skeletal muscle of young versus aged mice

Uncoupling protein 3 (UCP3) is suggested to protect mitochondria against aging and lipid-induced damage, possibly via modulation of reactive oxygen species (ROS) production. Here we show that mice overexpressing UCP3 (UCP3Tg) have a blunted age-induced increase in ROS production, assessed by electron spin resonance spectroscopy, but only after addition of 4-hydroxynonenal (4-HNE). Mitochondrial function, assessed by respirometry, on glycolytic substrate was lower in UCP3Tg mice compared to wild types, whereas this tended to be higher on fatty acids. State 4o respiration was higher in UCP3Tg animals. To conclude, UCP3 overexpression leads to increased state 4o respiration and, in presence of 4-HNE, blunts the age-induced increase in ROS production.

Local energetic regulation of sarcoplasmic and myosin ATPase is differently impaired in rats with heart failure

Local control of ATP/ADP ratio is essential for efficient functioning of cellular ATPases. Since creatine kinase (CK) activity and mitochondrial content are reduced in heart failure (HF), and cardiomyocyte ultrastructure is altered, we hypothesized that these changes may affect the local energetic control of two major cardiac ATPases, the sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA) and the myosin ATPase. Heart failure was induced by aortic stenosis in rats. Electron microscopy confirmed that failing cardiomyocytes had intracellular disorganization, with fewer contacts between mitochondria and myofibrils. Despite normal SERCA protein content, spontaneous Ca2+ release measurements using Fluo-4 on saponin-permeabilized cardiomyocytes showed a lower SR loading in HF even when endogenous CK and mitochondria were fully activated. Similarly, in permeabilized fibres, SR Ca2+ loading supported by SR-bound CK and mitochondria was significantly reduced in HF (by 49% and 40%, respectively, 43% when both systems were activated, P < 0.05). Alkaline phosphatase treatment had no effect, but glycolytic substrates normalized calcium loading in HF to the sham level. The control by CK and mitochondria of the local ATP/ADP ratio close to the myosin ATPase (estimated by rigor tension) was also significantly impaired in HF fibres (by 32% and 46%, respectively). However, while the contributions of mitochondria and CK to local ATP regeneration were equally depressed in HF for the control of SERCA, mitochondrial contribution was more severely impaired than CK (P < 0.05) with respect to myofilament regulation. These data show that local energetic regulation of essential ATPases is severely impaired in heart failure, and undergoes a complex remodelling as a result of a decreased activity of the ATP-generating systems and cytoarchitecture disorganization.

Effects of Anoxia and Aglycemia on Cytosolic Calcium Regulation in Rat Sensory Neurons

Nociceptive neurons play an important role in ischemia by sensing and transmitting information to the CNS and by secreting peptides and nitric oxide, which can have local effects. While these responses are probably primarily mediated by acid sensing channels, other events occurring in ischemia may also influence neuron function. In this study, we have investigated the effects of anoxia and anoxic aglycemia on Ca2+ regulation in sensory neurons from rat dorsal root ganglia. Anoxia increased [Ca2+]i by evoking Ca2+ release from two distinct internal stores one sensitive to carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) and one sensitive to caffeine, cyclopiazonic acid (CPA), and ryanodine [assumed to be the endoplasmic reticulum (ER)]. Anoxia also promoted progressive decline in ER Ca2+ content. Despite partially depolarizing mitochondria, anoxia had relatively little effect on mitochondrial Ca2+ uptake when neurons were depolarized but substantially delayed mitochondrial Ca2+ release and subsequent Ca2+ clearance from the cytosol on repolarization. Anoxia also reduced both sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) activity and Ca2+ extrusion [probably via plasma membrane Ca2+-ATPase (PMCA)]. Thus anoxia has multiple effects on [Ca2+]i homeostasis in sensory neurons involving internal stores, mitochondrial buffering, and Ca2+ pumps. Under conditions of anoxic aglycemia, there was a biphasic and more profound elevation of [Ca2+]i, which was associated with complete ER Ca2+ store emptying and progressive, and eventually complete, inhibition of Ca2+ clearance by PMCA and SERCA. These data clearly show that loss of oxygen, and exhaustion of glycolytic substrates, can profoundly affect many aspects of cell Ca2+ regulation, and this may play an important role in modulating neuronal responses to ischemia.

DNA polymerase γ inhibition by vitamin K3 induces mitochondria‐mediated cytotoxicity in human cancer cells

Among the vitamin K (VK) compounds, VK3 exhibits distinct cytotoxic activity in cancer cells and is thought to affect redox cycling; however, the underlying mechanisms remain unclear. Here we demonstrate that VK3 selectively inhibits DNA polymerase (pol) gamma, the key enzyme responsible for mitochondrial DNA replication and repair. VK3 at 30 microM inhibited pol gamma by more than 80%, caused impairment of mitochondrial DNA replication and repair, and induced a significant increase in reactive oxygen species (ROS), leading to apoptosis. At a lower concentration (3 microM), VK3 did not cause a significant increase in ROS, but was able to effectively inhibit cell proliferation, which could be reversed by supplementing glycolytic substrates. The cytotoxic action of VK3 was independent of p53 tumor suppressor gene status. Interestingly, VK3 only inhibited pol gamma but did not affect other pol including human pol alpha, pol beta, pol delta, and pol epsilon. VK1 and VK2 exhibited no inhibitory effect on any of the pol tested. These data together suggest that the inhibition of pol gamma by VK3 is relatively specific, and that this compound seems to exert its anticancer activity by two possible mechanisms in a concentration-dependent manner: (1) induction of ROS-mediated cell death at high concentrations; and (2) inhibition of cell proliferation at lower concentrations likely through the suppression of mitochondrial respiratory function. These findings may explain various cytotoxic actions induced by VK3, and may pave the way for the further use of VK3.

Role of apoptosis in acetaminophen hepatotoxicity

Acetaminophen overdose causes liver injury by mechanisms involving glutathione depletion, oxidative stress and mitochondrial dysfunction. The role of apoptosis in acetaminophen-induced cell killing is still controversial. Here, our aim was to evaluate the mitochondrial permeability transition (MPT) as a key factor in acetaminophen-induced necrotic and apoptotic killing of primary cultured mouse hepatocytes. Acetaminophen (10 micromol/L) induced necrotic killing in approximately 50% of hepatocytes after 6 h and cyclosporin A (CsA), MPT inhibitor, temporarily decreased necrotic killing after 6 h, but cytoprotection was lost after 16 h. Confocal microscopy revealed mitochondrial depolarization and inner membrane permeabilization at approximately 4.5 h after acetaminophen. CsA delayed these changes indicative of the MPT to about 11 h after acetaminophen. TUNEL labeling and caspase 3 activation also increased after acetaminophen. Fructose (20 mmol/L, an ATP-generating glycolytic substrate) plus glycine (5 mmol/L, a membrane stabilizing amino acid) prevented nearly all necrotic cell killing but paradoxically increased apoptosis. In conclusion, acetaminophen induces the MPT and ATP-depletion-dependent necrosis or caspase-dependent apoptosis as determined, in part, by ATP availability from glycolysis.

Implications and Treatment of Acute Hyperglycemia in the Setting of Acute Myocardial Infarction

A 52-year-old obese male without a prior history of diabetes mellitus (DM) presented with angina and an anterior ST-segment–elevation myocardial infarction (STEMI). Physical examination and chest x-ray were consistent with congestive heart failure. Admission glucose was 230 mg/dL. Coronary angiography revealed an occluded left anterior descending coronary artery, and stenting reestablished TIMI grade 2 flow in that artery within 90 minutes of symptom onset. Left ventricular ejection fraction was 35% with severe anterior hypokinesis. Peak creatine kinase was 600 IU. The next day, fasting glucose was 180 mg/dL. An echocardiogram performed 6 weeks after discharge revealed an ejection fraction of 35% without change in the anterior wall motion. Fasting glucose as an outpatient was 156 mg/dL. The scenario described above is commonly encountered and illustrates how hyperglycemia can affect the outcome of patients with STEMI. Hyperglycemia could have affected the following features of this case: (1) Congestive heart failure was present despite only modest myocardial injury by creatine kinase level; (2) despite successful percutaneous coronary intervention, subnormal coronary perfusion was observed; and (3) left ventricular recovery after STEMI did not occur. Cardiologists need to be cognizant of the hazards associated with hyperglycemia in this setting because these patients will be encountered more frequently as a result of the increasing prevalence of insulin resistance syndromes. Acute hyperglycemia is common in patients with STEMI even in the absence of a history of type 2 DM. Hyperglycemia is encountered in up to 50% of all STEMI patients, whereas previously diagnosed DM is present in only 20% to 25% of STEMI patients.1 The prevalence of type 2 DM or impaired glucose tolerance may be as high as 65% in MI patients without prior DM when oral glucose tolerance testing is performed.2 Elevated …

Roles of Hemoglobin Allostery in Hypoxia-induced Metabolic Alterations in Erythrocytes: SIMULATION AND ITS VERIFICATION BY METABOLOME ANALYSIS

When erythrocytes are exposed to hypoxia, hemoglobin (Hb) stabilizes in the T-state by capturing 2,3-bisphosphoglycerate. This process could reduce the intracellular pool of glycolytic substrates, jeopardizing cellular energetics. Recent observations suggest that hypoxia-induced activation of glycolytic enzymes is correlated with their release from Band III (BIII) on the cell membrane. Based on these data, we developed a mathematical model of erythrocyte metabolism and compared hypoxia-induced differences in predicted activities of the enzymes, their products, and cellular energetics between models with and without the interaction of Hb with BIII. The models predicted that the allostery-dependent Hb interaction with BIII accelerates consumption of upstream glycolytic substrates such as glucose 6-phosphate and increases downstream products such as phosphoenolpyruvate. This prediction was consistent with metabolomic data from capillary electrophoresis mass spectrometry. The hypoxia-induced alterations in the metabolites resulted from acceleration of glycolysis, as judged by increased conversion of [(13)C]glucose to [(13)C]lactate. The allostery-dependent interaction of Hb with BIII appeared to contribute not only to maintenance of energy charge but also to further synthesis of 2,3-bisphosphoglycerate, which could help sustain stabilization of T-state Hb during hypoxia. Furthermore, such an activation of glycolysis was not observed when Hb was stabilized in R-state by treating the cells with CO. These results suggest that Hb allostery in erythrocytes serves as an O(2)-sensing trigger that drives glycolytic acceleration to stabilize intracellular energetics and promote the ability to release O(2) from the cells.

Glycogen formation in Corynebacterium glutamicum and role of ADP-glucose pyrophosphorylase

Glycogen is generally assumed to serve as a major reserve polysaccharide in bacteria. In this work, glycogen accumulation in the amino acid producer Corynebacterium glutamicum was characterized, expression of the C. glutamicum glgC gene, encoding the key enzyme in glycogen synthesis, ADP-glucose (ADP-Glc) pyrophosphorylase, was analysed, and the relevance of this enzyme for growth, survival, amino acid production and osmoprotection was investigated. C. glutamicum cells grown in medium containing the glycolytic substrates glucose, sucrose or fructose showed rapid glycogen accumulation (up to 90 mg per g dry weight) in the early exponential growth phase and degradation of the polymer when the sugar became limiting. In contrast, no glycogen was detected in cells grown on the gluconeogenic substrates acetate or lactate. In accordance with these results, the specific activity of ADP-Glc pyrophosphorylase was 20-fold higher in glucose-grown than in acetate- or lactate-grown cells. Expression analysis suggested that this carbon-source-dependent regulation might be only partly due to transcriptional control of the glgC gene. Inactivation of the chromosomal glgC gene led to the absence of ADP-Glc pyrophosphorylase activity, to a complete loss of intracellular glycogen in all media tested and to a distinct lag phase when the cells were inoculated in minimal medium containing 750 mM sodium chloride. However, the growth of C. glutamicum, its survival in the stationary phase and its glutamate and lysine production were not affected by glgC inactivation under either condition tested. These results indicate that intracellular glycogen formation is not essential for growth and survival of and amino acid production by C. glutamicum and that ADP-Glc pyrophosphorylase activity might be advantageous for fast adaptation of C. glutamicum to hyperosmotic stress.

ATP-generating glycolytic substrates prevent N-nitrosofenfluramine-induced cytotoxicity in isolated rat hepatocytes

The relationship between cytotoxicity induced by N-nitrosofenfluramine and mitochondrial or glycolytic adenosine triphosphate (ATP) synthesis-dependent intracellular bioenergetics was studied in isolated rat hepatocytes. The supplementation of fructose, an ATP-generating glycolytic substrate, to hepatocyte suspensions prevented N-nitrosofenfluramine-induced cell injury accompanied by the formation of cell blebs, abrupt loss of intracellular ATP and reduced glutathione and mitochondrial membrane potential (DeltaPsi), and the accumulation of oxidized glutathione and malondialdehyde, indicating lipid peroxidation, during a 2h incubation period. Fructose (1-20mM) resulted in concentration-dependent protection against the cytotoxicity of N-nitrosofenfluramine at a concentration of 0.6mM, a low toxic dose. Pretreatment with xylitol, another glycolytic substrate, at concentration of 15mM also prevented the cytotoxicity caused by the nitroso compound, but neither glucose nor sucrose exhibited protective effects. In addition, fructose inhibited N-nitrosofenfluramine (0.5 and 0.6mM)-induced DNA damage, as evaluated in the comet assay, indicating that nuclei as well as mitochondria are target sites of the compound. These results indicate that (a) the onset of N-nitrosofenfluramine-induced cytotoxicity in rat hepatocytes is linked to mitochondrial failure, and that (b) the insufficient supply of ATP in turn limits the activities of all energy-requiring reactions and consequently leads to acute cell death.