Copper Accelerates Glycolytic Flux in Cultured Astrocytes

Astrocyte-rich primary cultures were used to investigate the consequences of a copper exposure on the glucose metabolism of astrocytes. After application of CuCl₂ (30 μM) the specific cellular copper content increased from initial 1.5 ± 0.2 nmol/mg to a steady state level of 7.9 ± 0.9 nmol/mg within about 12 h. The copper accumulation was accompanied by a significant increase in the extracellular lactate concentration. The stimulating effect of copper on the lactate production remained after removal of extracellular copper. Copper treatment accelerated the rates of both glucose consumption and lactate production by about 60%. The copper induced acceleration of glycolytic flux was prevented by inhibition of protein synthesis, and additive to the stimulation of glycolysis observed for inhibitors of respiration or prolyl hydroxylases. A copper induced stimulation of glycolytic flux in astrocytes could have severe consequences for the glucose metabolism of the brain in conditions of copper overload.     

Control of Glycolytic Flux by AMP-Activated Protein Kinase in Tumor Cells Adapted to Low pH

Tumor cells grow in nutrient- and oxygen-deprived microenvironments and adapt to the suboptimal growth conditions by altering their metabolic pathways. This adaptation process commonly results in a tumor phenotype that displays a high rate of aerobic glycolysis and aggressive tumor characteristics. The glucose regulatory molecule, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), is a bifunctional enzyme that is central to glycolytic flux and is downstream of the metabolic stress sensor AMP-activated protein kinase (AMPK), which has been suggested to modulate glycolysis and possibly activate isoforms of PFKFB, specifically PFKFB3 expressed in tumor cells. Our results demonstrated that long-term low pH exposure induced AMPK activation, which resulted in the up-regulation of PFKFB3 and an increase in its serine residue phosphorylation. Pharmacologic activation of AMPK resulted in an increase in PFKFB3 as well as an increase in glucose consumption, whereas in contrast, inhibition of AMPK resulted in the down-regulation of PFKFB3 and decreased glycolysis. PFKFB3 overexpression in DB-1 tumor cells induced a high rate of glycolysis and inhibited oxygen consumption, confirming its role in controlling glycolytic flux. These results show that low pH is a physiological stress that can promote a glycolytic phenotype commonly associated with tumorigenesis. The implications are that the tumor microenviroment contributes to tumor growth and treatment resistance.     

QSOX1 Inhibits Autophagic Flux in Breast Cancer Cells

The QSOX1 protein (Quiescin Sulfhydryl oxidase 1) catalyzes the formation of disulfide bonds and is involved in the folding and stability of proteins. More recently, QSOX1 has been associated with tumorigenesis and protection against cellular stress. It has been demonstrated in our laboratory that QSOX1 reduces proliferation, migration and invasion of breast cancer cells in vitro and reduces tumor growth in vivo. In addition, QSOX1 expression has been shown to be induced by oxidative or ER stress and to prevent cell death linked to these stressors. Given the function of QSOX1 in these two processes, which have been previously linked to autophagy, we wondered whether QSOX1 might be regulated by autophagy inducers and play a role in this catabolic process. To answer this question, we used in vitro models of breast cancer cells in which QSOX1 was overexpressed (MCF-7) or extinguished (MDA-MB-231). We first showed that QSOX1 expression is induced following amino acid starvation and maintains cellular homeostasis. Our results also indicated that QSOX1 inhibits autophagy through the inhibition of autophagosome/lysosome fusion. Moreover, we demonstrated that inhibitors of autophagy mimic the effect of QSOX1 on cell invasion, suggesting that its role in this process is linked to the autophagy pathway. Previously published data demonstrated that extinction of QSOX1 promotes tumor growth in NOG mice. In this study, we further demonstrated that QSOX1 null tumors present lower levels of the p62 protein. Altogether, our results demonstrate for the first time a role of QSOX1 in autophagy in breast cancer cells and tumors.     

A Rapid and Specific Microplate Assay for the Determination of Intra- and Extracellular Ascorbate in Cultured Cells

Vitamin C (ascorbate) plays numerous important roles in cellular metabolism, many of which have only come to light in recent years. For instance, within the brain, ascorbate acts in a neuroprotective and neuromodulatory manner that involves ascorbate cycling between neurons and vicinal astrocytes - a relationship that appears to be crucial for brain ascorbate homeostasis. Additionally, emerging evidence strongly suggests that ascorbate has a greatly expanded role in regulating cellular and systemic iron metabolism than is classically recognized. The increasing recognition of the integral role of ascorbate in normal and deregulated cellular and organismal physiology demands a range of medium-throughput and high-sensitivity analytic techniques that can be executed without the need for highly expensive specialist equipment. Here we provide explicit instructions for a medium-throughput, specific and relatively inexpensive microplate assay for the determination of both intra- and extracellular ascorbate in cell culture.     

Quantification of Rare Cancer Cells in Patients With Gastrointestinal Cancer by Nanostructured Substrate

Detecting the cancer cells in the peripheral blood, i.e. circulating tumor cell (CTC), have been considered as the “liquid biopsy” and become a particular area of focus. A deep insight into CTC provides a potential alternative method for early diagnosis of solid tumor. Previous studies showed that CTC counts could be regarded as an indicator in tumor diagnosis, predicting clinical outcomes and monitoring treatment responses. In this report, we utilize our facile and efficient CTC detection device made of hydroxyapatite/chitosan (HA/CTS) for rare cancer cells isolation and enumeration in clinical use. A biocompatible and surface roughness controllable nanofilm was deposited onto a glass slide to achieve enhanced topographic interactions with nanoscale cellular surface components, anti-EpCAM (epithelial cell adhesion molecule, EpCAM) were then coated onto the surface of nanosubstrate for specific capture of CTCs. This device performed a considerable and stable capture yields. We evaluated the relationship performance between serial CTC changes and the changes of tumor volume/serum tumor marker in gastrointestinal cancer patients undergoing anti-cancer treatments. The present study results showed that changes in the number of CTC were associated with tumor burden and progression. Enumeration of CTCs in cancer patients may predict clinical response. Longitudinal monitoring of individual patients during the therapeutic process showed a close correlation between CTC quantity and clinical response to anti-cancer therapy. Effectively capture of this device is capable of CTCs isolation and quantification for monitoring of cancer and predicting treatment response.     

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.     

Combination of Sulindac and Dichloroacetate Kills Cancer Cells via Oxidative Damage

Sulindac is an FDA-approved non-steroidal anti-inflammatory drug with documented anticancer activities. Our recent studies showed that sulindac selectively enhanced the killing of cancer cells exposed to oxidizing agents via production of reactive oxygen species (ROS) resulting in mitochondrial dysfunction. This effect of sulindac and oxidative stress on cancer cells could be related to the defect in respiration in cancer cells, first described by Warburg 50 years ago, known as the Warburg effect. We postulated that sulindac might enhance the selective killing of cancer cells when combined with any compound that alters mitochondrial respiration. To test this hypothesis we have used dichloroacetate (DCA), which is known to shift pyruvate metabolism away from lactic acid formation to respiration. One might expect that DCA, since it stimulates aerobic metabolism, could stress mitochondrial respiration in cancer cells, which would result in enhanced killing in the presence of sulindac. In this study, we have shown that the combination of sulindac and DCA enhances the selective killing of A549 and SCC25 cancer cells under the conditions used. As predicted, the mechanism of killing involves ROS production, mitochondrial dysfunction, JNK signaling and death by apoptosis. Our results suggest that the sulindac-DCA drug combination may provide an effective cancer therapy.     

High Glycolytic Flux Improves Pyruvate Production by a Metabolically Engineered Escherichia coli Strain

We report pyruvate formation in Escherichia coli strain ALS929 containing mutations in the aceEF, pfl, poxB, pps, and ldhA genes which encode, respectively, the pyruvate dehydrogenase complex, pyruvate formate lyase, pyruvate oxidase, phosphoenolpyruvate synthase, and lactate dehydrogenase. The glycolytic rate and pyruvate productivity were compared using glucose-, acetate-, nitrogen-, or phosphorus-limited chemostats at a growth rate of 0.15 h⁻¹. Of these four nutrient limitation conditions, growth under acetate limitation resulted in the highest glycolytic flux (1.60 g/g · h), pyruvate formation rate (1.11 g/g · h), and pyruvate yield (0.70 g/g). Additional mutations in atpFH and arcA (strain ALS1059) further elevated the steady-state glycolytic flux to 2.38 g/g · h in an acetate-limited chemostat, with heterologous NADH oxidase expression causing only modest additional improvement. A fed-batch process with strain ALS1059 using defined medium with 5 mM betaine as osmoprotectant and an exponential feeding rate of 0.15 h⁻¹ achieved 90 g/liter pyruvate, with an overall productivity of 2.1 g/liter · h and yield of 0.68 g/g.     

Inhibition of Oxidative Phosphorylation Induces a Rapid Death of GA-Pretreated Aleurone Cells,But Not of ABA-Pretreated Aleurone Cells

Reactive oxygen species (ROS) mediate programmed cell death in aleurone cells, which is promoted by gibberellic acid (GA) and prevented by abscisic acid (ABA). Plant mitochondria contain two distinct respiratory pathways: respiration through cytochrome c oxidase increases ROS production, whereas respiration through the alternative oxidase pathway lowers it. While studying the effects of GA and ABA on partitioning of respiration between those two pathways during the germinating process, we discovered that oxidative phosphorylation inhibitors like sodium azide and 2, 4-dinitrophenol induce rapid death of GA-pretreated aleurone cells but not of ABA-pretreated cells. Functional aerobic respiration was required for GA signaling, and 6 to 12 hours of GA signaling altered the cellular state of aleurone cells to be extremely susceptible to inhibition of oxidative phosphorylation. Anaerobic conditions were also able to mimic the effects of respiratory inhibitors in specifically inducing cell death in GA-treated cells, but cell death was provoked much more slowly. Cotreatment with various antioxidants did not prevent this process at all, suggesting that no ROS are responsible for this respiratory inhibitor-induced cell death. Our observation implicates that GA may partition all the electrons produced during mitochondrial respiration only to the cytochrome oxidase pathway, which would at least partly contribute to cellular accumulation of ROS.     

The Plasma Membrane Calcium Pump in Pancreatic Cancer Cells Exhibiting the Warburg Effect Relies on Glycolytic ATP

Evidence suggests that the plasma membrane Ca²⁺-ATPase (PMCA), which is critical for maintaining a low intracellular Ca²⁺ concentration ([Ca²⁺]_i), utilizes glycolytically derived ATP in pancreatic ductal adenocarcinoma (PDAC) and that inhibition of glycolysis in PDAC cell lines results in ATP depletion, PMCA inhibition, and an irreversible [Ca²⁺]_i overload. We explored whether this is a specific weakness of highly glycolytic PDAC by shifting PDAC cell (MIA PaCa-2 and PANC-1) metabolism from a highly glycolytic phenotype toward mitochondrial metabolism and assessing the effects of mitochondrial versus glycolytic inhibitors on ATP depletion, PMCA inhibition, and [Ca²⁺]_i overload. The highly glycolytic phenotype of these cells was first reversed by depriving MIA PaCa-2 and PANC-1 cells of glucose and supplementing with α-ketoisocaproate or galactose. These culture conditions resulted in a significant decrease in both glycolytic flux and proliferation rate, and conferred resistance to ATP depletion by glycolytic inhibition while sensitizing cells to mitochondrial inhibition. Moreover, in direct contrast to cells exhibiting a high glycolytic rate, glycolytic inhibition had no effect on PMCA activity and resting [Ca²⁺]_i in α-ketoisocaproate- and galactose-cultured cells, suggesting that the glycolytic dependence of the PMCA is a specific vulnerability of PDAC cells exhibiting the Warburg phenotype.     

Sulindac Enhances the Killing of Cancer Cells Exposed to Oxidative Stress

Background

Sulindac is an FDA-approved non-steroidal anti-inflammatory drug (NSAID) that affects prostaglandin production by inhibiting cyclooxygenases (COX) 1 and 2. Sulindac has also been of interest for more than decade as a chemopreventive for adenomatous colorectal polyps and colon cancer.

Principal Findings

Pretreatment of human colon and lung cancer cells with sulindac enhances killing by an oxidizing agent such as tert-butyl hydroperoxide (TBHP) or hydrogen peroxide. This effect does not involve cyclooxygenase (COX) inhibition. However, under the conditions used, there is a significant increase in reactive oxygen species (ROS) within the cancer cells and a loss of mitochondrial membrane potential, suggesting that cell death is due to apoptosis, which was confirmed by Tunel assay. In contrast, this enhanced killing was not observed with normal lung or colon cells.

Significance

These results indicate that normal and cancer cells handle oxidative stress in different ways and sulindac can enhance this difference. The combination of sulindac and an oxidizing agent could have therapeutic value.     

Glycolytic Flux and Hexokinase Activities in Anoxic Maize Root Tips Acclimated by Hypoxic Pretreatment

In vitro cyclic electron transport around PSI was studied in thylakoids isolated from barley (Hordeum vulgare L.). Redox poising was obtained by using anaerobic conditions, preillumination, and the addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Postillumination rates of P700+ re-reduction of 1 to 5 electrons s-1 were observed, depending on the conditions. The thylakoids supported two parallel paths of cyclic electron transport that were distinguishable by differences in antimycin sensitivity, saturation characteristics, and substrate specificity. The pathway most sensitive to antimycin was not saturated at ferredoxin concentrations up to 50 [mu]M, whereas the more insensitive pathway was saturated at 5 [mu]M ferredoxin. At the lower concentration of reduced ferredoxin, the antimycin-sensitive rate of P700+ re-reduction was lower than the antimycin-insensitive rate. The lower range of reduced ferredoxin concentrations are closer to in vivo conditions. Flavodoxin is shown to mediate cyclic electron transport. Flavodoxin was less efficient in mediating the antimycin-sensitive pathway but mediated the antimycin-insensitive pathway as efficiently as ferredoxin. Antibodies raised against ferredoxin:NADP+ oxidoreductase had no effect on either pathway for re-reduction of P700+. However, the ferredoxin: NADP+ oxidoreductase inhibitor 2[prime]-monophosphoadenosine-5[prime]-diphosphoribose was able to inhibit the antimycin-sensitive as well as the antimycin-insensitive pathway.     

Consumption of NADPH for 2-HG Synthesis Increases Pentose Phosphate Pathway Flux and Sensitizes Cells to Oxidative Stress

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Glycolytic Flux Signals to mTOR through Glyceraldehyde-3-Phosphate Dehydrogenase-Mediated Regulation of Rheb

The mammalian target of rapamycin (mTOR) interacts with raptor to form the protein complex mTORC1 (mTOR complex 1), which plays a central role in the regulation of cell growth in response to environmental cues. Given that glucose is a primary fuel source and a biosynthetic precursor, how mTORC1 signaling is coordinated with glucose metabolism has been an important question. Here, we found that the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) binds Rheb and inhibits mTORC1 signaling. Under low-glucose conditions, GAPDH prevents Rheb from binding to mTOR and thereby inhibits mTORC1 signaling. High glycolytic flux suppresses the interaction between GAPDH and Rheb and thus allows Rheb to activate mTORC1. Silencing of GAPDH or blocking of the Rheb-GAPDH interaction desensitizes mTORC1 signaling to changes in the level of glucose. The GAPDH-dependent regulation of mTORC1 in response to glucose availability occurred even in TSC1-deficient cells and AMPK-silenced cells, supporting the idea that the GAPDH-Rheb pathway functions independently of the AMPK axis. Furthermore, we show that glyceraldehyde-3-phosphate, a glycolytic intermediate that binds GAPDH, destabilizes the Rheb-GAPDH interaction even under low-glucose conditions, explaining how high-glucose flux suppresses the interaction and activates mTORC1 signaling. Taken together, our results suggest that the glycolytic flux regulates mTOR''s access to Rheb by regulating the Rheb-GAPDH interaction, thereby allowing mTORC1 to coordinate cell growth with glucose availability.The mTOR complex 1 (mTORC1) signal transduction pathway acts as a central controller of cell growth in mammals (20, 23, 29). mTORC1 integrates a wide range of intracellular and extracellular signals, including insulin, availability of nutrients (glucose and amino acids), cellular energy status, and hypoxia, to regulate protein synthesis and cell growth (11, 12, 17, 36, 46). Many of these environmental cues are integrated into tuberous sclerosis complex (TSC1-TSC2), the major upstream regulator of mTORC1. In response to the absence of insulin and to the low-energy status of cells, the TSC1-TSC2 complex stimulates the GTPase function of Rheb, a small GTPase that acts as a proximal key activator of mTORC1, which leads to the inhibition of Rheb-mediated mTORC1 activation. In contrast, inactivation of the TSC1-TSC2 complex results in the accumulation of GTP-bound Rheb and thus activation of mTORC1 (3, 13, 21, 27, 32, 39). For this reason, both the loss of TSC proteins and the overexpression of Rheb cause hyperactivation of mTORC1 signaling, which is frequently observed in many common human cancers (2, 5, 19, 25, 33). Therefore, a tight regulation of Rheb activity is critical for the proper operation of the mTORC1 pathway in response to environmental cues.Rheb is an atypical member of the Ras superfamily of GTPases (1, 10, 47). As with other small GTPases, the activity of Rheb is regulated by its guanine nucleotide binding status. However, the negative control of GTP-bound Rheb by the TSC1-TSC2 complex has only recently been investigated, and the regulation of the nucleotide binding status of Rheb is not fully understood. A recent study proposed that translationally controlled tumor protein may function as a guanine nucleotide exchange factor for Rheb that causes the accumulation of GTP-bound Rheb (18). GTP-bound Rheb is essential for activating mTOR kinase (21, 28, 38). However, the interaction between Rheb and mTOR does not depend on the GTP binding status of Rheb (30), raising questions regarding the mechanism by which Rheb activates mTORC1. Recently, FKBP38 (immunophilin FK506-binding protein, 38 kDa) was found to be a direct binding partner of Rheb and an inhibitor of mTORC1 (4). GTP-bound Rheb binds FKBP38 and releases FKBP38 from mTORC1, resulting in activation of the mTORC1 pathway. However, there have been conflicting results regarding the effects of nutrient availability on Rheb activity (31, 37, 42, 50) and the effect of these newly identified regulators of Rheb function (44, 45). Thus, the precise molecular mechanisms underlying Rheb regulation and Rheb-mediated mTORC1 activation have remained unclear.In this study, we identified glyceraldehyde-3-phosphate (Gly-3-P) dehydrogenase (GAPDH) as a novel Rheb binding protein and a negative regulator of Rheb. We found that the interaction between GAPDH and Rheb is induced when the glycolytic flux is suppressed under low-glucose conditions to inhibit mTORC1. Here, we provide a molecular mechanism underlying the cross talk between the glycolytic flux and the mTORC1 signaling.     

Oxidative and Glycolytic Recovery Metabolism in Muscle : Fluorometric observations on their relative contributions

The average degree of reduction of mitochondrial NAD has been measured in the intact toad sartorius by a fluorometric technique. It has been shown that cytoplasmic NADH does not interfere materially with these measurements. The percentage reduction of this respiratory coenzyme has been determined in a number of physiological steady states which are well correlated with fluorometrically determined levels of NADH in suspensions of mitochondria from the hind leg musculature of the toad. In addition, these findings are closely comparable to similar, spectrophotometric measurements on mitochondria from other sources. In the presence of an adequate O₂ level a single twitch produces a decrease in fluorescence from the resting steady state which is followed by a slow return to the base line condition. This cycle indicates the intensity and the time course of the oxidative recovery metabolism. The area under this curve is directly related to the number of twitches up to three or four. Greater activity produces a curtailment of oxidative recovery due to glycolysis. In the presence of iodoacetate the linear relation holds for five to seven twitches. At still higher levels of activity a curtailment of the change in NAD level sets in, probably due to the removal of AMP by catabolic reactions.     

Oxidative Stress Induces Nuclear-to-Cytosol Shift of hMSH3, a Potential Mechanism for EMAST in Colorectal Cancer Cells

Background

Elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) is a genetic signature observed in 60% of sporadic colorectal cancers (CRCs). Unlike microsatellite unstable CRCs where hypermethylation of the DNA mismatch repair (MMR) gene hMLH1’s promoter is causal, the precise cause of EMAST is not clearly defined but points towards hMSH3 deficiency.

Aim

To examine if hMSH3 deficiency causes EMAST, and to explore mechanisms for its deficiency.

Methods

We measured −4 bp framshifts at D8S321 and D20S82 loci within EGFP-containing constructs to determine EMAST formation in MMR-proficient, hMLH1^−/−, hMSH6^−/−, and hMSH3^−/− CRC cells. We observed the subcellular location of hMSH3 with oxidative stress.

Results

D8S321 mutations occurred 31-and 40-fold higher and D20S82 mutations occurred 82-and 49-fold higher in hMLH1^−/− and hMSH3^−/− cells, respectively, than in hMSH6^−/− or MMR-proficient cells. hMSH3 knockdown in MMR-proficient cells caused higher D8S321 mutation rates (18.14 and 11.14×10⁻⁴ mutations/cell/generation in two independent clones) than scrambled controls (0 and 0.26×10⁻⁴ mutations/cell/generation; p<0.01). DNA sequencing confirmed the expected frameshift mutations with evidence for ongoing mutations of the constructs. Because EMAST-positive tumors are associated with inflammation, we subjected MMR-proficient cells to oxidative stress via H₂O₂ to examine its effect on hMSH3. A reversible nuclear-to-cytosol shift of hMSH3 was observed upon H₂O₂ treatment.

Conclusion

EMAST is dependent upon the MMR background, with hMSH3^−/− more prone to frameshift mutations than hMSH6^−/−, opposite to frameshift mutations observed for mononucleotide repeats. hMSH3^−/− mimics complete MMR failure (hMLH1^−/−) in inducing EMAST. Given the observed heterogeneous expression of hMSH3 in CRCs with EMAST, hMSH3-deficiency appears to be the event that commences EMAST. Oxidative stress, which causes a shift of hMSH3’s subcellular location, may contribute to an hMSH3 loss-of-function phenotype by sequestering it to the cytosol.     

Rapid In Vitro Derivation of Endothelium Directly From Human Cancer Cells

The development of an independent blood supply by a tumor is essential for maintaining growth beyond a certain limited size and for providing a portal for metastatic dissemination. Host-derived endothelial cells (ECs) residing in and compromising the tumor vasculature originate via distinct processes known as sprouting angiogenesis and vasculogenesis. More recently ECs originating directly from the tumor cells themselves have been described although the basis for this phenomenon remains poorly understood. Here we describe in vitro conditions that allow lung and ovarian cancer cells to undergo a rapid and efficient transition into ECs that are indistinguishable from those obtained in vivo. A variety of methods were used to establish that the acquired phenotypes and behaviors of these tumor-derived ECs (TDECs) closely resemble those of authentic ECs. Xenografts arising from co-inoculated in vitro-derived TDECs and tumor cells were also more highly vascularized than control tumors; moreover, their blood vessels were on average larger and frequently contained admixtures of host-derived ECs and TDECs derived from the initial inoculum. These results demonstrate that cancer cells can be manipulated under well-defined in vitro conditions to initiate a tumor cell-to-EC transition that is largely cell-autonomous, highly efficient and closely mimics the in vivo process. These studies provide a suitable means by which to identify and perhaps modify the earliest steps in TDEC generation.     

Evidence for a Large and Sustained Glycolytic Flux to Lactate in Anoxic Roots of Some Members of the Halophytic Genus Limonium

Soil salinity and anaerobiosis often occur together. This led us to investigate the fermentative metabolism in roots of species from the halophytic genus Limonium (Plumbaginaceae). Root segments from hypoxically induced plants were incubated for 8 h under strict anoxia in the presence of [U-14C]glucose. In three species (Limonium latifolium, L. nashii, and L. humile), the pattern of 14C-labeled end products was typical of higher plants, with a 14C flux to ethanol higher than that to lactate. However, in four species (L. ramosissimum, L. gougetianum, L perezii, and L. sinuatum), the rate of lactate fermentation was exceptionally high, and in the latter two species the 14C flux to lactate exceeded that to ethanol. These two species secreted most of the lactate produced into the medium. Calculations indicated that the cytoplasm would have been lethally acidified had this secretion not occurred. The effects of factors that might control lactate fermentation or secretion (O2 partial pressure, pH, salt concentration) were studied in two contrasting species: L. sinuatum and L. latifolium. In both species, the lactate:ethanol ratio was higher under hypoxia (0.1-3 kPa O2 partial pressure) than under strict anoxia. In L. sinuatum, this ratio was slightly increased by increasing the pH of the medium from 5.5 to 7.5, but salinity treatment had no effect. The potential contribution of lactate fermentation to the overall carbon and energy metabolism of halophytes is discussed.