The tricarboxylic acid cycle and glycolysis in relation to ion transport by the ciliary body

1. The respiration and aerobic glycolysis of pig ciliary processes in oxygenated phosphate and bicarbonate buffers have been investigated. 2. Significant amounts of lactic acid are produced only in the presence of added glucose, but this does not change the endogenous respiration rate. 3. Succinate and citrate increase the oxygen uptake considerably, but pyruvate has almost no effect; oxaloacetate and fumarate stimulate slightly in the presence of glucose. Aspartate and fumarate together stimulate pyruvate utilization and are oxidized as fast as citrate. 4. Ouabain inhibits the oxidation of glucose and other substrates by limiting the ADP supply from the sodium transport system. Cyanide and azide inhibit respiration and stimulate glycolysis. 5. The transport mechanism depends largely on ATP from oxidative phosphorylation and regulates the rate of respiration and glycolysis by controlling ADP production from the Na(+)-K(+)-activated adenosine triphosphatase.     

Berberine improves glucose metabolism through induction of glycolysis

Berberine, a botanical alkaloid used to control blood glucose in type 2 diabetes in China, has recently been reported to activate AMPK. However, it is not clear how AMPK is activated by berberine. In this study, activity and action mechanism of berberine were investigated in vivo and in vitro. In dietary obese rats, berberine increased insulin sensitivity after 5-wk administration. Fasting insulin and HOMA-IR were decreased by 46 and 48%, respectively, in the rats. In cell lines including 3T3-L1 adipocytes, L6 myotubes, C2C12 myotubes, and H4IIE hepatocytes, berberine was found to increase glucose consumption, 2-deoxyglucose uptake, and to a less degree 3-O-methylglucose (3-OMG) uptake independently of insulin. The insulin-induced glucose uptake was enhanced by berberine in the absence of change in IRS-1 (Ser307/312), Akt, p70 S6, and ERK phosphorylation. AMPK phosphorylation was increased by berberine at 0.5 h, and the increase remained for > or =16 h. Aerobic and anaerobic respiration were determined to understand the mechanism of berberine action. The long-lasting phosphorylation of AMPK was associated with persistent elevation in AMP/ATP ratio and reduction in oxygen consumption. An increase in glycolysis was observed with a rise in lactic acid production. Berberine exhibited no cytotoxicity, and it protected plasma membrane in L6 myotubes in the cell culture. These results suggest that berberine enhances glucose metabolism by stimulation of glycolysis, which is related to inhibition of glucose oxidation in mitochondria. Berberine-induced AMPK activation is likely a consequence of mitochondria inhibition that increases the AMP/ATP ratio.     

Aerobic and anaerobic carbohydrate metabolism and egg production of Schistosoma mansoni in vitro.

When S. mansoni adults were cultured in vitro for 12 days in a diphasic medium, their gross morphology, motor activity, frequency of sexual pairings, rates of glucose utilization and of lactic acid production were the same in the presence (90% N2/5% O2/5% CO2) or absence (95% N2/5% CO2) of oxygen. Therefore, no Pasteur effect, nor any reduction in lactic acid formation, was demonstrable under aerobic conditions. While aerobic conditions did not affect the rate of glycolysis, they had a marked effect on egg production. In the presence of oxygen, the rate of egg-laying reached a maximum between days 4 and 6. The average number of viable eggs produced per worm pair during this period was 118 (Sx equals 2.2), which is within the overall range (68 to 248) recorded by others for this same strain in vivo. Conversely, under anaerobic conditions in vitro, virtually no eggs were laid. It remains to be determined whether oxidative metabolism actually is required for energy to produce eggs, or whether some reaction yielding no ATP is essential for completion of their developmental process, such as tanning of the eggshall brought about by the oxidation of some phenolic compounds.     

THE EFFECT OF LOW OXYGEN TENSION ON TISSUE METABOLISM (RETINA)

Lactic acid production by rat retina in a medium containing phosphate was studied chemically. One half as much lactic acid was found as in a medium containing bicarbonate. In our experience the rate of respiration in a phosphate medium was sensitive to oxygen tension, for it was 38 per cent lower at 10 per cent and 51 per cent lower at 5 per cent oxygen than at 100 per cent oxygen. Previously Laser had reported no decrease in respiration at 5 per cent oxygen in phosphate medium. In phosphate medium, when the oxygen tension was varied, respiration and glycolysis bore a reciprocal relationship to each other. In bicarbonate medium, when the oxygen tension was lowered from 95 per cent to 5 per cent there was no significant change in the respiration, but glycolysis was increased nearly to the anaerobic level. This agrees with the earlier experiment of Laser in bicarbonate medium and adds support to his conclusion that the rate of glycolysis is controlled by oxygen tension rather than by the rate of respiration, under the conditions of the experiment.     

Studies on brain-cortex slices: The influence of various inhibitors on the retention of potassium ions and amino acids with glucose or pyruvate as substrate

1. The rate of appearance of (14)CO(2) from [6-(14)C]glucose and [3-(14)C]pyruvate was measured. Pyruvate is oxidized to carbon dioxide twice as fast as glucose, although the oxygen uptake is almost the same with each substrate. 2. The presence of 30mum-2,4-dinitrophenol increases the output of (14)CO(2) from [6-(14)C]glucose sixfold whereas the oxygen uptake is not quite doubled. Similar results are obtained with 0.1m-potassium chloride. The stimulating action of these two agents on the output of (14)CO(2) from [3-(14)C]pyruvate is much less than on that from [6-(14)C]glucose. 3. The effects of oligomycin, ouabain and triethyltin on the respiration of control and stimulated brain-cortex slices were studied. Triethyltin (1.3mum) inhibited the oxidation of [6-(14)C]glucose more than 70%, but did not inhibit the oxidation of[3-(14)C]pyruvate. [3-(14)C]pyruvate. 4. The production of lactic acid by brain-cortex slices incubated with glucose is twice as great as that with pyruvate. Lactic acid increases two and a half times in the presence of either triethyltin or oligomycin when the substrate is glucose, but is no different from the control when the substrate is pyruvate. 5. With kidney slices the production of lactic acid from glucose is very low. It is increased by oligomycin but not by triethyltin. 6. The results are discussed in terms of the oxidation of the extramitochondrial NADH(2) produced during glycolysis.     

Schistosoma mansoni: biochemical characterization of lactate transporters or similar proteins

While in medium containing glucose, schistosomes exhibit homolactic fermentation. Accumulation of lactate acid in tissue fluid causes lowering of pH and a resultant inhibition of metabolic pathways. This requires lactate transporter protein in homolactic fermentors to facilitate the translocation of lactate(-) and [H(+)] across their plasma membrane. The ex-vivo experiment assessed lactic acid secretion by adult worms in absence and the presence of lactic acid transporter protein inhibitors. Phloretin and alpha-cyano-4-hydroxycinnamate caused a combined 25-35% inhibition of lactic acid secretion and probenecid increased this inhibition to 65% of control values. The removal of inhibitors resulted in 80% recovery of lactic acid secretion. In the in-vitro studies using vesicles isolated from adult worms and from schistosomula, the effects of phloretin and alpha-cyano-4-hydroxycinnamate were greater, each causing approximately 80% inhibition independently. The data obtained in this study demonstrate the presence of lactic acid transporters or similar proteins in Schistosoma mansoni.     

Glutamine and glucose as energy substrates for Ehrlich ascites tumour cells

Energy metabolism of freshly harvested Ehrlich ascites tumour cells in the presence of 5 mM glucose and/or 0.5 mM glutamine was studied. The rate of oxygen utilization was not altered by the addition of 0.5 mM glutamine; 5 mM glucose induced an inhibition of respiration. In the presence of both glucose and glutamine, the Crabtree effect decreased. In these conditions, the rates of oxygen uptake, the CO2 evolution and the changes in the redox states of cytochromes indicate that glucose is preferred by Ehrlich ascites tumour cells as energy substrate. Glucose decreased the rate of glutamine utilization by 34%. On the other hand, glutaminolysis did not inhibit glycolysis.     

Effect of additive solutions on platelet biochemistry.

Use of a synthetic medium for resuspension and storage of platelets has several advantages. In addition to the opportunity to save plasma, the use of a synthetic medium offers better possibilities for obtaining platelet products by automatic component processing in a more cost-effective and convenient manner. A synthetic medium also has the potential to avoid transfusion reactions caused by noncompatible plasma proteins and to improve platelet quality, which will lessen the number of units needed in transfusion. Platelets produce a substantial amount of lactic acid by metabolism of glucose. Thus, risk of pH fall is a major problem which platelet additive solutions must address. Two approaches have been used: (1) use of a glucose-free medium, and (2) use of a glucose-containing medium with a buffer system to neutralize the production of lactic acid. Using various synthetic media described in the literature, studies described in this paper suggest that some glucose is needed in the final suspending medium in order to obtain satisfactory maintenance of platelet quality during 5 days of storage. With the presence of acetate in the additive solution, platelet respiration is increased and glycolysis substantially decreased; therefore, with 10-20% of CPD-plasma carryover, sufficient glucose may be present in the final suspending medium to last for 5 days of storage. In addition, phosphate in the additive solution markedly enhances platelet glycolysis, and inhibitors of platelet activation reduce platelet metabolic activity and improve platelet viability and function.     

Glutamine‐driven oxidative phosphorylation is a major ATP source in transformed mammalian cells in both normoxia and hypoxia

Mammalian cells can generate ATP via glycolysis or mitochondrial respiration. Oncogene activation and hypoxia promote glycolysis and lactate secretion. The significance of these metabolic changes to ATP production remains however ill defined. Here, we integrate LC‐MS‐based isotope tracer studies with oxygen uptake measurements in a quantitative redox‐balanced metabolic flux model of mammalian cellular metabolism. We then apply this approach to assess the impact of Ras and Akt activation and hypoxia on energy metabolism. Both oncogene activation and hypoxia induce roughly a twofold increase in glycolytic flux. Ras activation and hypoxia also strongly decrease glucose oxidation. Oxidative phosphorylation, powered substantially by glutamine‐driven TCA turning, however, persists and accounts for the majority of ATP production. Consistent with this, in all cases, pharmacological inhibition of oxidative phosphorylation markedly reduces energy charge, and glutamine but not glucose removal markedly lowers oxygen uptake. Thus, glutamine‐driven oxidative phosphorylation is a major means of ATP production even in hypoxic cancer cells.     

Oncosecretomics coupled to bioenergetics identifies α-amino adipic acid, isoleucine and GABA as potential biomarkers of cancer: Differential expression of c-Myc, Oct1 and KLF4 coordinates metabolic changes

Bioenergetic profiling of tumors is a new challenge of cancer research and medicine as therapies are currently being developed. Meanwhile, methodological means must be proposed to gather information on tumor metabolism in order to adapt these potential therapies to the bioenergetic specificities of tumors. Studies performed on tumors and cancer cell lines have shown that cancer cells bioenergetics is highly variable. This profile changes with microenvironmental conditions (eg. substrate availability), the oncogenes activated (and the tumor suppressors inactivated) and the interaction with the stroma (i.e. reverse Warburg effect). Here, we assessed the power of metabolic footprinting (MFP) to unravel the bioenergetics and associated anabolic changes induced by three oncogenes, c-Myc, KLF4 and Oct1. The MFP approach provides a quantitative analysis of the metabolites secreted and consumed by cancer cells. We used ultra performance liquid chromatography for quantifying the amino acid uptake and secretion. To investigate the potential oncogene-mediated alterations in mitochondrial metabolism, we measured oxygen consumption rate and ATP production as well as the glucose uptake and lactate release. Our findings show that c-Myc deficiency initiates the Warburg effect along with a reduction of mitochondrial respiration. KLF4 deficiency also stimulated glycolysis, albeit without cellular respiration impairment. In contrast, Oct1 deficiency reduced glycolysis and enhanced oxidative phosphorylation efficiency. MFP revealed that c-Myc, KLF4 and Oct1 altered amino acid metabolism with specific patterns. We identified isoleucine, α-aminoadipic acid and GABA (γ-aminoisobutyric acid) as biomarkers related. Our findings establish the impact of Oct1, KLF4 and c-Myc on cancer bioenergetics and evidence a link between oncosecretomics and cellular bioenergetics profile.     

Overexpression of Mitochondrial Sirtuins Alters Glycolysis and Mitochondrial Function in HEK293 Cells

SIRT3, SIRT4, and SIRT5 are mitochondrial deacylases that impact multiple facets of energy metabolism and mitochondrial function. SIRT3 activates several mitochondrial enzymes, SIRT4 represses its targets, and SIRT5 has been shown to both activate and repress mitochondrial enzymes. To gain insight into the relative effects of the mitochondrial sirtuins in governing mitochondrial energy metabolism, SIRT3, SIRT4, and SIRT5 overexpressing HEK293 cells were directly compared. When grown under standard cell culture conditions (25 mM glucose) all three sirtuins induced increases in mitochondrial respiration, glycolysis, and glucose oxidation, but with no change in growth rate or in steady-state ATP concentration. Increased proton leak, as evidenced by oxygen consumption in the presence of oligomycin, appeared to explain much of the increase in basal oxygen utilization. Growth in 5 mM glucose normalized the elevations in basal oxygen consumption, proton leak, and glycolysis in all sirtuin over-expressing cells. While the above effects were common to all three mitochondrial sirtuins, some differences between the SIRT3, SIRT4, and SIRT5 expressing cells were noted. Only SIRT3 overexpression affected fatty acid metabolism, and only SIRT4 overexpression altered superoxide levels and mitochondrial membrane potential. We conclude that all three mitochondrial sirtuins can promote increased mitochondrial respiration and cellular metabolism. SIRT3, SIRT4, and SIRT5 appear to respond to excess glucose by inducing a coordinated increase of glycolysis and respiration, with the excess energy dissipated via proton leak.     

Glucose uptake and lactic acid production of adenovirus type 5-infected HEp-2 cells cultured under exponential growth and stationary phase conditions

Exponentially growing HEp-2 cells have a higher rate of glucose uptake and lactic acid production than stationary phase cells. Infection of cells with adenovirus type 5 stimulates glycolysis irrespective of the original rate of the host cells. Therefore, infected cells cultured under exponential growth conditions have a higher rate of glycolysis than infected stationary phase cells. The rate of host cell glycolysis does not influence the time required for virus replication, the yield of infectious virus particles produced, or the time of appearance and progression of virus-induced cytopathology.     

微RNA在肿瘤细胞糖代谢中的功能

大多数癌细胞产生能量是通过高速率糖酵解,然后在胞液中进行乳酸发酵。而在大多数正常细胞中,糖酵解速率相对较低,丙酮酸主要在线粒体中进行有氧氧化。即使在氧充足的条件下,快速生长的恶性肿瘤细胞进行糖酵解的速率通常要比其正常组织来源的细胞高二百多倍。微RNA（microRNA,miRNA）是一类具有转录后调控功能的非编码RNA。近年来,越来越多的研究表明,miRNA主要通过诱导缺氧环境、影响葡萄糖摄入、调节糖酵解过程中的关键酶以及乳酸去路等诸多方面参与糖代谢过程,从而在肿瘤细胞糖代谢中发挥重要作用。     

Melatonin Cytotoxicity Is Associated to Warburg Effect Inhibition in Ewing Sarcoma Cells

Melatonin kills or inhibits the proliferation of different cancer cell types, and this is associated with an increase or a decrease in reactive oxygen species, respectively. Intracellular oxidants originate mainly from oxidative metabolism, and cancer cells frequently show alterations in this metabolic pathway, such as the Warburg effect (aerobic glycolysis). Thus, we hypothesized that melatonin could also regulate differentially oxidative metabolism in cells where it is cytotoxic (Ewing sarcoma cells) and in cells where it inhibits proliferation (chondrosarcoma cells). Ewing sarcoma cells but not chondrosarcoma cells showed a metabolic profile consistent with aerobic glycolysis, i.e. increased glucose uptake, LDH activity, lactate production and HIF-1α activation. Melatonin reversed Ewing sarcoma metabolic profile and this effect was associated with its cytotoxicity. The differential regulation of metabolism by melatonin could explain why the hormone is harmless for a wide spectrum of normal and only a few tumoral cells, while it kills specific tumor cell types.     

A global view of the biochemical pathways involved in the regulation of the metabolism of cancer cells

Cancer cells increase glucose uptake and reject lactic acid even in the presence of oxygen (Warburg effect). This metabolism reorients glucose towards the pentose phosphate pathway for ribose synthesis and consumes great amounts of glutamine to sustain nucleotide and fatty acid synthesis. Oxygenated and hypoxic cells cooperate and use their environment in a manner that promotes their development. Coenzymes (NAD(+), NADPH,H(+)) are required in abundance, whereas continuous consumption of ATP and citrate precludes the negative feedback of these molecules on glycolysis, a regulation supporting the Pasteur effect. Understanding the metabolism of cancer cells may help to develop new anti-cancer treatments.     

Anaerobic shift of energy metabolism in the mouse brain during the recovery period in acute radiation sickness

Three months after whole-body irradiation of mice with a sublethal dose of 5 Gy a study was made of some indices of energy metabolism like tissue respiration, oxidative phosphorylation, and formation of lactic acid in the survived brain homogenate. Revealed were the diminution of coupling of tissue respiration of oxidative phosphorylation, the rate of oxygen consumption and the level of cyano-resistant respiration being constant, the increase in the rate of glycolysis in anaerobic and particularly, in aerobic conditions, and reduction of the Pasteur and Crabtree effects. The above mentioned changes in the brain energy metabolism seem to be a manifestation of the process of the reduced metabolism formation in the nervous tissue at the remote times after irradiation.     

ASPP2 suppresses tumour growth and stemness characteristics in HCC by inhibiting Warburg effect via WNT/β-catenin/HK2 axis

Abnormal energy metabolism is one of the characteristics of tumours. In the last few years, more and more attention is being paid to the role and regulation of tumour aerobic glycolysis. Cancer cells display enhanced aerobic glycolysis, also known as the Warburg effect, whereby tumour cells absorb glucose to produce a large amount of lactic acid and energy under aerobic conditions to favour tumour proliferation and metastasis. In this study, we report that the haploinsufficient tumour suppressor ASPP2, can inhibit HCC growth and stemness characteristics by regulating the Warburg effect through the WNT/β-catenin pathway. we performed glucose uptake, lactate production, pyruvate production, ECAR and OCR assays to verify ASPP2 can inhibit glycolysis in HCC cells. The expression of ASPP2 and HK2 was significantly inversely correlated in 80 HCC tissues. Our study reveals downregulation of ASPP2 can promote the aerobic glycolysis metabolism pathway, increasing HCC proliferation, glycolysis metabolism, stemness and drug resistance. This ASPP2-induced inhibition of glycolysis metabolism depends on the WNT/β-catenin pathway. ASPP2-regulated Warburg effect is associated with tumour progression and provides prognostic value. and suggest that may be promising as a new therapeutic strategy in HCC.     

Role of hexose transport in control of glycolytic flux in Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae predominantly ferments glucose to ethanol at high external glucose concentrations, irrespective of the presence of oxygen. In contrast, at low external glucose concentrations and in the presence of oxygen, as in a glucose-limited chemostat, no ethanol is produced. The importance of the external glucose concentration suggests a central role for the affinity and maximal transport rates of yeast's glucose transporters in the control of ethanol production. Here we present a series of strains producing functional chimeras between the hexose transporters Hxt1 and Hxt7, each of which has distinct glucose transport characteristics. The strains display a range of decreasing glycolytic rates resulting in a proportional decrease in ethanol production. Using these strains, we show for the first time that at high glucose levels, the glucose uptake capacity of wild-type S. cerevisiae does not control glycolytic flux during exponential batch growth. In contrast, our chimeric Hxt transporters control the rate of glycolysis to a high degree. Strains whose glucose uptake is mediated by these chimeric transporters will undoubtedly provide a powerful tool with which to examine in detail the mechanism underlying the switch between fermentation and respiration in S. cerevisiae and will provide new tools for the control of industrial fermentations.