GPER促进乳腺癌相关成纤维细胞的有氧糖酵解

为探讨GPER基因的活化对人乳腺癌相关成纤维细胞(CAF)有氧糖酵解的影响,本研究构建GPERsi RNA慢病毒,转染CAF细胞,以构建GPER敲低的稳定细胞系;对照组(CAF-shNC)和GPER-shRNA慢病毒感染组(CAF-shGPER组),经嘌呤霉素筛选后,实时荧光定量PCR和蛋白印迹法检测GPER的m RNA及蛋白的表达;用GPER特异性激动剂G1 (1μmol/L)处理以上两组细胞,得到G1+CAF-shNC和G1+CAFsh GPER,应用蛋白印迹法分别检测以上4组细胞中GPER下游基因p-PKA和p-CREB的表达;采用实时荧光定量PCR和蛋白印迹法检测CREB糖酵解相关靶基因PDK4和LDHB的表达,应用葡萄糖、乳酸检测试剂盒检测4组细胞中葡萄糖消耗,乳酸生成情况。最终,成功构建GPER敲低的CAF稳定细胞系;GPER特异性激动剂G1可明显上调CAF-shNC组中GPER mRNA和蛋白水平;G1+CAF-shNC与CAF-shNC和G1+CAFsh GPER组相比,GPER下游基因PKA和CREB的磷酸化水平显著增加(p<0.05);CREB糖酵解相关靶基因PDK4和LDHB的mRNA和蛋白水平明显上调(p<0.05);葡萄糖消耗量和乳酸生成量明显增加(p<0.05)。由此可得,在人乳腺癌相关成纤维细胞中,GPER特异性激动剂G1活化GPER促进CAF的有氧糖酵解。     

Aerobic Glycolysis by Proliferating Cells: Protection against Oxidative Stress at the Expense of Energy Yield

Primary cultures of mitogen-activated rat thymocytes were used to study energy metabolism, gene expression of glycolytic enzymes, and production of reactive oxygen species during cell cycle progression. During transition from the resting to the proliferating state a 7- to 10-fold increase of glycolytic enzyme induction occurs which enables the cells to meet the enhanced energy demand by increased aerobic glycolysis. Cellular redox changes have been found to regulate gene expression of glycolytic enzymes by reversible oxidative inactivation of Spl-binding to the cognate DNA-binding sites in the promoter region. In contrast to nonproliferating cells, production of phorbol 12-myristate 13-acetate (PMA)-primed reactive oxygen species (ROS) in proliferating rat thymocytes and HL-60 cells is nearly abolished. Pyruvate, a product of aerobic glycolysis, is an effective scavenger of ROS, which could be shown to be generated mainly at the site of complex III of the mitochondrial respiratory chain. Aerobic glycolysis by proliferating cells is discussed as a means to minimize oxidative stress during the phases of the cell cycle when maximally enhanced biosynthesis and cell division do occur.     

Kinetic Parameters and Lactate Dehydrogenase Isozyme Activities Support Possible Lactate Utilization by Neurons

Lactate is potentially a major energy source in brain, particularly following hypoxia/ischemia; however, the regulation of brain lactate metabolism is not well understood. Lactate dehydrogenase (LDH) isozymes in cytosol from primary cultures of neurons and astrocytes, and freshly isolated synaptic terminals (synaptosomes) from adult rat brain were separated by electrophoresis, visualized with an activity-based stain, and quantified. The activity and kinetics of LDH were determined in the same preparations. In synaptosomes, the forward reaction (pyruvate + NADH + H⁺ → lactate + NAD⁺), which had a V _max of 1,163 μmol/min/mg protein was 62% of the rate in astrocyte cytoplasm. In contrast, the reverse reaction (lactate + NAD⁺ → pyruvate + NADH + H⁺), which had a V _max of 268 μmol/min/mg protein was 237% of the rate in astrocytes. Although the relative distribution was different, all five isozymes of LDH were present in synaptosomes and primary cultures of cortical neurons and astrocytes from rat brain. LDH1 was 14.1% of the isozyme in synaptic terminals, but only 2.6% and 2.4% in neurons and astrocytes, respectively. LDH5 was considerably lower in synaptic terminals than in neurons and astrocytes, representing 20.4%, 37.3% and 34.8% of the isozyme in these preparations, respectively. The distribution of LDH isozymes in primary cultures of cortical neurons does not directly reflect the kinetics of LDH and the capacity for lactate oxidation. However, the kinetics of LDH in brain are consistent with the possible release of lactate by astrocytes and oxidative use of lactate for energy in synaptic terminals. Special issue dedicated to John P. Blass.     

Aerobic expression of Vitreoscilla hemoglobin improves the growth performance of CHO‐K1 cells

Inefficient carbon metabolism is a relevant issue during the culture of mammalian cells for the production of biopharmaceuticals. Therefore, cell engineering strategies to improve the metabolic and growth performance of cell lines are needed. The expression of Vitreoscilla stercoraria hemoglobin (VHb) has been shown to significantly reduce overflow metabolism and improve the aerobic growth of bacteria. However, the effects of VHb on mammalian cells have been rarely studied. Here, the impact of VHb on growth and lactate accumulation during CHO‐K1 cell culture was investigated. For this purpose, CHO‐K1 cells were transfected with plasmids carrying the vgb or gfp gene to express VHb or green fluorescence protein (GFP), respectively. VHb expression increased the specific growth rate and biomass yields on glucose and glutamine by 60 %, and reduced the amount of lactate produced per cell by 40 %, compared to the GFP‐expression controls. Immunofluorescence microscopy showed that VHb is distributed in the cytoplasm and organelles, which support the hypothesis that VHb could serve as an oxygen carrier, enhancing aerobic respiration. These results are useful for the development of better producing cell lines for industrial applications.     

Revisiting the Crabtree/Warburg effect in a dynamic perspective: a fitness advantage against sugar-induced cell death

The mechanisms behind the Warburg effect in mammalian cells, as well as for the similar Crabtree effect in the yeast Saccharomyces cerevisiae, are still a matter of debate: why do cells shift from the energy-efficient respiration to the energy-inefficient fermentation at high sugar concentration?

This review reports on the strong similarities of these phenomena in both cell types, discusses the current ideas, and provides a novel interpretation of their common functional mechanism in a dynamic perspective. This is achieved by analysing another phenomenon, the sugar-induced-cell-death (SICD) occurring in yeast at high sugar concentration, to highlight the link between ATP depletion and cell death.

The integration between SICD and the dynamic functioning of the glycolytic process, suggests that the Crabtree/Warburg effect may be interpreted as the avoidance of ATP depletion in those conditions where glucose uptake is higher than the downstream processing capability of the second phase of glycolysis. It follows that the down-regulation of respiration is strategic for cell survival allowing the allocation of more resources to the fermentation pathway, thus maintaining the cell energetic homeostasis.     

Substrates of Energy Metabolism of the Pituitary and Pineal Glands

The capability of the neurohypophysis, the adenohypophysis, and the pineal gland to oxidize nonesterified fatty acids and glucose as energy sources was studied in vivo. Fed and 48-h-starved rats had catheters placed in their femoral vessels. After they became conscious, an intravenous injection of one of the following was given: [1-14C]acetate, [1-14C]octanoate, [1-14C]-palmitate, or [2-14C]glucose. After 5 min the rats were sacrificed. These metabolites produce [14C]acetyl-CoA within the mitochondria when they are oxidized as metabolic fuels. On passage through the Krebs cycle a considerable portion of the 14C is trapped in large amino acid pools closely associated with the Krebs cycle; the appearance of 14C in these amino acids was taken as evidence of oxidation. As expected, brain structures behind the blood-brain barrier (cerebral cortex and caudate) showed considerable labeling of Krebs cycle-associated amino acids in both nutritional states when [2-14C]glucose was the substrate. Surprisingly, however, no label was detected in amino acids of the neurohypophysis or the pineal gland in starved rats and very little in fed rats. On the other hand, 14C from acetate and palmitate was extensively incorporated into amino acids of the pineal gland and the neurohypophysis, while little 14C labeling was found in the cerebral cortex and the caudate. Octanoate, which passes the blood-brain barrier readily, labeled amino acids of all tissues. The experiments demonstrated conclusively that the neural structures studied, which have no blood-brain barrier, do not rely heavily upon glucose as a fuel for oxidative energy metabolism, in contrast to the rest of the brain. The results also showed that nonesterified fatty acids may supply at least some of their energy requirements.     

Uptake and metabolism of pyruvate and glucose by individual sheep preattachment embryos developed in vivo

The uptake of pyruvate and glucose by individual sheep oocytes and preattachment sheep embryos at each state of development up to the hatching blastocyst was determined using a microfluorescence technique. After an initial increase at fertilization, pyruvate uptake was relatively constant (?15 pmol/embryo/h) from the zygote through to the morula. Upon blastocyst formation and hatching, there were significant increases in uptake (39 pmol/embryo/h, P < 0.001; and 53 pmol/embryo/h, P < 0.001, respectively). In contrast to that of pyruvate, glucose uptake was very low (?1 pmol/embryo/h) up to the time of genome activation (eight- to 16 cell stage), after which there were significant increases in uptake at each successive stage of development. By the hatching blastocyst stage, glucose uptake had reached 54 pmol/embryo/h. The ability of day-7 hatching blastocysts to oxidize pyruvate and glucose was determined indirectly by measuring the production of lactate when either substrate was present as the sole energy source. Unlike the mouse blastocyst, which has a considerable oxidative capacity for both pyruvate and glucose, the day-7 sheep blastocyst showed limited ability to oxidise either substrate. Rather, in the sheep blastocyst, 65% of pyruvate and 98% of glucose taken up could be accounted for as lactate. Such low levels of substrate oxidation appear to be inconsistent with the energy requirements of the proliferating preattachment ruminant blastocyst. The utilization of alternative substrates at the blastocyst, such as amino acids, is proposed. © 1993 Wiley-Liss, Inc.     

ATP-Sensitive Potassium Channels and Local Energy Demands in the Rat Hippocampus: An In Vivo Study

Abstract: Microdialysis coupled with an enzyme-based flow injection analysis was used to monitor brain extracellular lactate and glucose in the freely moving rat. Glucose levels reflect the balance between supply from the blood and local utilisation, and lactate efflux indicates the degree of local nonoxidative glucose metabolism. Local application of tolbutamide, a blocker of the ATP-sensitive potassium channel, decreased extracellular glucose and lactate levels in the hippocampus but not in the striatum. The increase in glucose and lactate levels following mild behavioural stimulation was also reduced by tolbutamide in the hippocampus. Similar effects on both basal and stimulated lactate levels were obtained with local application of 10 m M glucose. These results indicate that ATP-sensitive potassium channels are active under physiological conditions in the hippocampus and that the effects of tolbutamide can be mimicked by physiological glucose levels.     

Involvement of d-lactate and lactic acid racemase in the metabolism of glucose by Selenomonas ruminantium

Selenomonas ruminantium HD4 produced significant quantities of d- and l-lactate from glucose in batch culture. Both isomers also supported growth if fumarate was present. In glucose-limited continuous culture, d-lactate was detected in the medium only at fast dilution rates. In continuous-culture-grown cells, only a cytoplasmic NAD-dependent l-lactate dehydrogenase (LDH) and a membrane-associated NAD-independent l-LDH were detected; activity of the soluble enzyme was twice as high at the fast dilution rate as at the slow dilution rate. Lactate racemase was also detected; its activity was 4-fold higher at the fast dilution rate. The presence of racemase explains why d-lactate was made and used by this organism.     

Aerobic sugar metabolism in the spoilage yeast Zygosaccharomyces bailii

Despite the importance of some Zygosaccharomyces species as agents causing spoilage of food, the carbon and energy metabolism of most of them is yet largely unknown. This is the case with Zygosaccharomyces bailii. In this study the occurrence of the Crabtree effect in the petite-negative yeast Z. bailii ATCC 36947 was investigated. In this yeast the aerobic ethanol production is strictly dependent on the carbon source utilised. In glucose-limited continuous cultures a very low level of ethanol was produced. In fructose-limited continuous cultures ethanol was produced at a higher level and its production increased with the dilution rate. As a consequence, on fructose the onset of respiro-fermentative metabolism caused a reduction in biomass yield. An immediate aerobic alcoholic fermentation in Z. bailii was observed during the transition from sugar limitation to sugar excess, both on glucose and on fructose. The analysis of some key enzymes of the fermentative metabolism showed a high level of acetyl-CoA synthetase in Z. bailii growing on fructose. At high dilution rates, the activities of glucose- and fructose-phosphorylating enzymes, as well as of pyruvate decarboxylase and alcohol dehydrogenase, were higher in cells during growth on fructose than on glucose.     

Brain Lactate Is an Obligatory Aerobic Energy Substrate for Functional Recovery After Hypoxia: Further In Vitro Validation

Abstract: This study used the rat hippocampal slice preparation and the monocarboxylate transporter inhibitor, α-cyano-4-hydroxycinnamate (4-CIN), to assess the obligatory role that lactate plays in fueling the recovery of synaptic function after hypoxia upon reoxygenation. At a concentration of 500 µ M , 4-CIN blocked lactate-supported synaptic function in hippocampal slices under normoxic conditions in 15 min. The inhibitor had no effect on glucose-supported synaptic function. Of control hippocampal slices exposed to 10-min hypoxia, 77.8 ± 6.8% recovered synaptic function after 30-min reoxygenation. Of slices supplemented with 500 µ M 4-CIN, only 15 ± 10.9% recovered synaptic function despite the large amount of lactate formed during the hypoxic period and the abundance of glucose present before, during, and after hypoxia. These results indicate that 4-CIN, when present during hypoxia and reoxygenation, blocks lactate transport from astrocytes, where the bulk of anaerobic lactate is formed, to neurons, where lactate is being utilized aerobically to support recovery of function after hypoxia. These results unequivocally validate that brain lactate is an obligatory aerobic energy substrate for posthypoxia recovery of function.     

Lactate Is Released and Taken Up by Isolated Rabbit Vagus Nerve During Aerobic Metabolism

Abstract: To determine if lactate is produced during aerobic metabolism in peripheral nerve, we incubated pieces of rabbit vagus nerve in oxygenated solution containing d -[U-¹⁴C]glucose while stimulating electrically. After 30 min, nearly all the radioactivity in metabolites in the nerve was in lactate, glucose 6-phosphate, glutamate, and aspartate. Much lactate was released to the bath: 8.2 pmol (µg dry wt)⁻¹ from the exogenous glucose and 14.2 pmol (µg dry wt)⁻¹ from endogenous substrates. Lactate release was not increased when bath P o ₂ was decreased, indicating that it did not come from anoxic tissue. When the bath contained [U-¹⁴C]lactate at a total concentration of 2.13 m M and 1 m M glucose, ¹⁴C was incorporated in CO₂ and glutamate. The initial rate of formation of CO₂ from bath lactate was more rapid than its formation from bath glucose. The results are most readily explained by the hypothesis that has been proposed for brain tissue in which glial cells supply lactate to neurons.     

Nondestructive Measurement of Retinal Glucose Transport and Consumption In Vivo Using NMR Spectroscopy

Abstract: The cellular events underlying various retinopathies are poorly understood but likely involve perturbation of retinal glucose metabolism. Current methods for assessing this metabolism are destructive, thus limiting longitudinal studies. We hypothesize that following an intravitreous injection, the clearance rate of a glucose analogue will be a nondestructive index of retinal glucose transport and metabolism in vivo. First, radiolabeled glucose analogues were injected into the vitreous. After 40 min, the dominant clearance path was posterior via the retina and was consistent with a facilitated transport mechanism. Next, either [6,6-²H₂]glucose or 3-deoxy-3-fluoro- d -glucose was injected into the vitreous of rabbit eyes, and the clearance rate of each analogue was determined over 40 min using, respectively, ²H or ¹⁹F NMR. These rates were interpreted as a function of the retinal glucose transport and consumption. From the NMR data, the rate of retinal glucose consumption was ∼16 times slower than the transport of glucose. These data demonstrate that NMR measurements of glucose analogue clearance rate from the vitreous can provide a nondestructive index of retinal glucose transport and consumption in vivo.     

The changes in the energy metabolism of human muscle induced by training

The biochemical effects of training programmes have been studied with a kinetic model of central metabolism, using enzyme activities and metabolite concentrations measured at rest and after 30 s maximum-intensity exercise, collected before and after long and short periods of training, which differed only by the duration of the rest intervals. After short periods of training the glycolytic flux at rest was three times higher than it had been before training, whereas during exercise the flux and energy consumption remained the same as before training. Long periods of training had less effect on the glycolytic flux at rest, but increased it in response to exercise, increasing the contribution of oxidative phosphorylation.     

Direct Measurement of Extracellular Lactate in the Human Hippocampus During Spontaneous Seizures

Abstract: The effect of clinical, spontaneous-onset seizures on extracellular fluid lactate was investigated by the method of lactography, the in vivo on-line measurement of lactate levels using microdialysis. Studies of experimental animals have suggested that generation of extracellular lactate as measured by microdialysis is an index of local glucose utilization and is dependent on the activity of neurons under physiological conditions. Patients with medically refractory complex partial epilepsy underwent stereo-tactic implantation of combination depth electrode/micro-dialysis probes into both hippocampi for 7–16 days. During spontaneous complex partial seizures with secondary generalization, extracellular lactate levels rose by 91 β 32%. Moreover, this increase persisted for 60–90 min. During a unilateral hippocampal seizure that did not propagate to the contralateral hippocampus, the increase in lactate content was restricted to the side of seizure activity. Between seizures, extracellular lactate levels correlated with the frequency of interictal spikes. In summary, these data suggest that brief clinical seizures increase nonoxidative glucose metabolism significantly as measured by the generation of extracellular lactate. Furthermore, the increase in extracellular lactate level is limited to the site of seizure activity. Lactate is transported extracellularly via a lactate/proton cotransporter; therefore, the rise in extracellular lactate level may mediate the drop in pH_o associated with seizure activity. As acidification of the extracellular compartment has an inhibitory effect on neuronal excitability, the rise in extracellular lactate content may be a mechanism of seizure arrest and postictal refractoriness. Moreover, extracellular lactate may also mediate the decreased seizure susceptibility associated with frequent interictal spikes.     

1-Methyl-4-(2''-Ethylphenyl)-1,2,3,6-Tetrahydropyridine- Induced Toxicity in PC12 Cells Is Enhanced by Preventing Glycolysis

The effects of 1-methyl-4-(2'-ethylphenyl)-1,2,3,6-tetrahydropyridine (2'Et-MPTP), 1-methyl-4-(2'-ethylphenyl)pyridinium (2'Et-MPP+), and the classic complex 1 inhibitor, rotenone, on toxicity as well as on rates of glucose use and lactate production were studied using the pheochromocytoma PC12 cell line. PC12 cells are neoplastic in nature and have a high rate of glycolysis accompanied by a large production of lactate and a low use of glucose carbon through the Krebs cycle. 1-Methyl-4-phenylpyridinium (MPP+) and analogues such as 2'Et-MPP+ are actively accumulated by mitochondrial preparations in vitro and block NADH dehydrogenase of complex 1. This blockade results in biochemical sequelae that are ultimately cytotoxic. In this study, untreated PC12 cells used glucose and concomitantly accumulated lactate in a time-dependent manner at all concentrations of glucose studied. Treatment with 50 microM 2'Et-MPP+ or 50 nM rotenone increased both rates significantly, indicating a shift toward increased glycolysis. Cell death caused by the neurotoxins was also time and concentration dependent and markedly enhanced by glucose depletion in the medium. The increase in 2'Et-MPTP-induced toxicity in low glucose-supplemented cells was not due to an increase in pyridinium formation from the tetrahydropyridine, but rather to the lack of glucose for glycolysis. Moreover, inhibition of glycolysis with 2-deoxyglucose or iodoacetic acid also enhanced the lethality of the neurotoxins to the cells. The data in this study provide additional support to the hypothesis that 2'Et-MPP+ or related analogues act to kill cells by inhibiting mitochondrial respiration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phylogenetic Analysis of Proteins Associated in the Four Major Energy Metabolism Systems: Photosynthesis,Aerobic Respiration,Denitrification, and Sulfur Respiration

The four electron transfer energy metabolism systems, photosynthesis, aerobic respiration, denitrification, and sulfur respiration, are thought to be evolutionarily related because of the similarity of electron transfer patterns and the existence of some homologous proteins. How these systems have evolved is elusive. We therefore conducted a comprehensive homology search using PSI-BLAST, and phylogenetic analyses were conducted for the three homologous groups (groups 1–3) based on multiple alignments of domains defined in the Pfam database. There are five electron transfer types important for catalytic reaction in group 1, and many proteins bind molybdenum. Deletions of two domains led to loss of the function of binding molybdenum and ferredoxin, and these deletions seem to be critical for the electron transfer pattern changes in group 1. Two types of electron transfer were found in group 2, and all its member proteins bind siroheme and ferredoxin. Insertion of the pyridine nucleotide disulfide oxidoreductase domain seemed to be the critical point for the electron transfer pattern change in this group. The proteins belonging to group 3 are all flavin enzymes, and they bind flavin adenine dinucleotide (FAD) or flavin mononucleotide (FMN). Types of electron transfer in this group are divergent, but there are two common characteristics. NAD(P)H works as an electron donor or acceptor, and FAD or FMN transfers electrons from/to NAD(P)H. Electron transfer functions might be added to these common characteristics by the addition of functional domains through the evolution of group 3 proteins. Based on the phylogenetic analyses in this study and previous studies, we inferred the phylogeny of the energy metabolism systems as follows: photosynthesis (and possibly aerobic respiration) and the sulfur/nitrogen assimilation system first diverged, then the sulfur/nitrogen dissimilation system was produced from the latter system.     

Metabolism of Lactate in the Rat Brain During the Early Neonatal Period

The metabolism of lactate in isolated cells from early neonatal rat brain has been studied. In these circumstances, lactate was mainly oxidized to CO2, although a significant portion was incorporated into lipids (78% sterols, 4% phosphatidylcholine, 2% phosphatidylethanolamine, and 1% phosphatidylserine). The rate of lactate incorporation into CO2 and lipids was higher than those found for glucose and 3-hydroxybutyrate. Lactate strongly inhibited glucose oxidation through the pyruvate dehydrogenase-catalyzed reaction and the tricarboxylic acid cycle while scarcely affecting glucose utilization by the pentose phosphate pathway. Lipogenesis from glucose was strongly inhibited by lactate without relevant changes in the rate of glycerol phosphate synthesis. These results suggest that lactate inhibits glucose utilization at the level of the pyruvate dehydrogenase-catalyzed reaction, which may be a mechanism to spare glucose for glycerol and NADPH synthesis. The effect of 3-hydroxybutyrate inhibiting lactate utilization only at high concentrations of 3-hydroxybutyrate suggests that before ketogenesis becomes active, lactate may be the major fuel for the neonatal brain. (-)-Hydroxycitrate and aminooxyacetate markedly inhibited lipogenesis from lactate, suggesting that the transfer of lactate carbons through the mitochondrial membrane is accomplished by the translocation of both citrate and N-acetylaspartate.     

Molecular intricacies of aerobic glycolysis in cancer: current insights into the classic metabolic phenotype

Aerobic glycolysis is the process of oxidation of glucose into pyruvate followed by lactate production under normoxic condition. Distinctive from its anaerobic counterpart (i.e. glycolysis that occurs under hypoxia), aerobic glycolysis is frequently witnessed in cancers, popularly known as the “Warburg effect”, and it is one of the earliest known evidences of metabolic alteration in neoplasms. Intracellularly, aerobic glycolysis circumvents mitochondrial oxidative phosphorylation (OxPhos), facilitating an increased rate of glucose hydrolysis. This in turn enables cancer cells to successfully compete with normal cells for glucose uptake in order to maintain uninterrupted growth. In addition, evading OxPhos mitigates excessive generation/accumulation of reactive oxygen species that otherwise may be deleterious to cells. Emerging data indicate that aerobic glycolysis in cancer also promotes glutaminolysis to satisfy the precursor requirements of certain biosynthetic processes (e.g. nucleic acids). Next, the metabolic intermediates of aerobic glycolysis also feed the pentose phosphate pathway (PPP) to facilitate macromolecular biosynthesis necessary for cancer cell growth and proliferation. Extracellularly, the extrusion of the end-product of aerobic glycolysis, i.e. lactate, alters the tumor microenvironment, and impacts cancer-associated cells. Collectively, accumulating data unequivocally demonstrate that aerobic glycolysis implicates myriad of molecular and functional processes to support cancer progression. This review, in the light of recent research, dissects the molecular intricacies of its regulation, and also deliberates the emerging paradigms to target aerobic glycolysis in cancer therapy.