Hemoglobin expression affects ethylene production in maize cell cultures.

The formation of ethylene under different O(2) concentrations and upon addition of nitric oxide (NO) donor, sodium nitroprusside (SNP), was determined using maize (Zea mays L.) cell lines over-expressing (Hb+) or down-regulating (Hb-) hypoxically inducible (class-1) hemoglobin (Hb). Under all treatments, ethylene levels in the Hb- line were 5 to 6.5 times the levels in Hb+ and four to five times the levels in the wild type. Low oxygen partial pressures impaired ethylene formation in maize cell suspension cultures. 1-Amino-cyclopropane-1-carboxylic acid (ACC) oxidase (E.C. 1.14.17.4) mRNA levels did not vary, either between lines or between treatments. There was, however, significantly enhanced ACC oxidase (ACO) activity in the Hb- line relative to the wild type and the Hb+ line. ACO activity in the Hb- line increased under hypoxic conditions and significantly increased upon treatment with NO under normoxic conditions. The results suggest that limiting class-1 hemoglobin protein synthesis increases ethylene formation in maize suspension cells, possibly via the modulation of NO levels.     

The haemoglobin/nitric oxide cycle: involvement in flooding stress and effects on hormone signalling

BACKGROUND: Class 1 haemoglobins (Hbs) are induced in plant cells under hypoxic conditions. They have a high affinity for oxygen, which is two orders of magnitude lower than that of cytochrome oxidase, permitting the utilization of oxygen by the molecule at extremely low oxygen concentrations. Their presence reduces the levels of nitric oxide (NO) that is produced from nitrate ion during hypoxia and improves the redox and energy status of the hypoxic cell. SCOPE: The mechanism by which Hb interacts with NO under hypoxic conditions in plants is examined, and the effects of Hb expression on metabolism and signal transduction are discussed. CONCLUSIONS: The accumulated evidence suggests that a metabolic pathway involving NO and Hb provides an alternative type of respiration to mitochondrial electron transport under limited oxygen. Hb in hypoxic plants acts as part of a soluble, terminal, NO dioxygenase system, yielding nitrate ion from the reaction of oxyHb with NO. NO is mainly formed due to anaerobic accumulation of nitrite. The overall reaction sequence, referred to as the Hb/NO cycle, consumes NADH and maintains ATP levels via an as yet unknown mechanism. Hb gene expression appears to influence signal transduction pathways, possibly through its effect on NO, as evidenced by phenotypic changes in normoxic Hb-varying transgenic plants. Ethylene levels are elevated when Hb gene expression is suppressed, which could be a factor leading to root aerenchyma formation during hypoxic stress.     

Hemoglobin and Hypoxic Acclimation in Maize Root Tips

Class 1 hemoglobins (Hbs) have a wide distribution in the plant kingdom and have been demonstrated in root, seed, stem, and leaf tissues. They are present at low concentrations in aerobic tissue, but their synthesis is rapidly induced by hypoxic stress. The pattern of expression of the maize Hb gene in roots of young maize plants exposed to hypoxia has been examined. Root Hb gene expression increased rapidly to a maximum within first two hours of hypoxia, then declining to prehypoxia levels within 48-h hypoxic exposure. Limiting oxygen supply to the roots by total plant immersion and darkness did not alter the time course of hemoglobin expression. Hb gene expression was about 20-fold higher in the stele than in the cortex of control, aerobically grown roots. Stele Hb expression increased about fourfold under hypoxic conditions, whereas its expression in the cortex increased about 60-fold. In these samples, alcohol dehydrogenase (Adh) gene expression increased about four- and ten fold in the stele and cortex, respectively. The effect of the state of the Hb on anoxic survival of maize root tips was assessed by exposing root tips to a carbon monoxide atmosphere to maximize the proportion of hemoglobin in the carbonmonoxy form. Carbon monoxide had no significant effect on the survival or the ATP levels in anoxic maize roots, regardless of whether they had been acclimated by exposure to a hypoxic pretreatment. This would suggest that the presence of oxyhemoglobin is not essential for the survival of anoxic root tips.     

Class-1 hemoglobins,nitrate and NO levels in anoxic maize cell-suspension cultures

Nitric oxide (NO) is a reactive gas involved in many biological processes of animals, plants and microbes. Previous work has demonstrated that NO is formed during hypoxia in alfalfa (Medicago sativa L.) root cultures and that the levels of NO detected are inversely related to the levels of expression of class-1 hemoglobin expressed in the tissue. The objectives of this study were: to examine whether NO is produced in transgenic maize (Zea mays L.) cell-suspension cultures exposed to anoxic growth conditions; to determine whether a similar relationship existed between a class-1 hemoglobin and the amount of NO detected under these conditions; and, to estimate the route of formation and breakdown of NO in the tissue. Maize cell-suspension cultures, transformed to express the sense or antisense strands of barley hemoglobin were used to overexpress or underexpress class-1 hemoglobin. A maize cell-suspension culture transformed with an empty vector was used as a control. Up to 500 nmol NO (g FW)^–1 was detected in maize cells exposed to low oxygen tensions for 24 h. The steady-state levels of NO in the different cell lines under anoxic conditions had an inverse relationship to the level of hemoglobin in the cells. There was no detectable NO produced under aerobic growth conditions. Spectroscopic data demonstrated that recombinant maize hemoglobin reacted with NO to form methemoglobin and NO₃^–. Nitrate was shown to be a precursor of NO in anoxic maize cell-suspension cultures by using ¹⁵NO₃^– and electron paramagnetic resonance spectroscopy, suggesting that NO is formed via nitrate reductase during hypoxia. The results demonstrate that NO is produced in plant tissues grown under low oxygen tensions and suggest that class-1 hemoglobins have a significant function in regulating NO levels.Abbreviations DEANO 2-(N,N-Diethylamino)-diazenolate-2-oxide - EPR Electron paramagnetic resonance - Hb Hemoglobin - MGD N-(Dithiocarbamoyl)-N-methyl-d-glucamine - NOS Nitric oxide synthase - WT Wild type     

Class-1 hemoglobin and antioxidant metabolism in alfalfa roots

In the course of nitric oxide (NO) scavenging, hemoglobin (Hb) turnover is linked to antioxidant metabolism and affects the cellular redox level. The influence of Hb presence on the ascorbate-glutathione cycle enzymes and the levels of H₂O₂ and ascorbate was investigated in alfalfa root cultures transformed to over-express (Hb+) or down-regulate (Hb–) class-1 Hb. Hb+ lines had substantially increased ascorbate levels as well as elevated monodehydroascorbate reductase and ascorbate peroxidase activities. Hb– lines showed significant increases in dehydroascorbate reductase and glutathione reductase activities. The observed changes in ascorbate and ascorbate-glutathione cycle enzymes were pronounced both at high (40 kPa) and low (3 kPa) O₂ pressures. Hb– lines had significantly reduced levels of the NO- and H₂O₂-sensitive enzyme, aconitase, as compared to Hb+ lines. This reduced activity was likely due the higher levels of NO in Hb– lines, as treatment of plant extracts with the NO-donor DEANO also affected aconitase activity. The H₂O₂ levels were not significantly different amongst the lines and showed no variation with change in oxygen partial pressure. In conclusion, the expression of class-1 Hb improves the antioxidant status through increased ascorbate levels and increased activity of enzymes involved in H₂O₂ removal.     

Intracellular adenosine triphosphate as a measure of human tumor cell viability and drug modulated growth

Summary Adenosine triphosphate is the primary energy unit for cells, and levels of this compound offer a potential marker for cell viability and growth. The availability of a bioluminescence assay allows for a rapid, sensitive, and reproducible measurement of ATP. A method is described for the quantification of intracellular ATP levels in human cancer cells. ATP levels were linearly related to the number of viable cells and increased with time in human cancer cell line cultures correlating with growth kinetics. The effect of 5-fluorouracil, doxorubicin, methotrexate, cytosine arabinoside, nitrogen mustard, melphalan, vinblastine, and cisplatin on the growth of human cancer cell lines was studied utilizing ATP levels. ATP levels and colony formation in agar of drug-exposed cells were compared. Overall there was a significant correlation between drug effects on colony formation and ATP levels. The ATP assay is rapid, simple, reproducible, and a relatively inexpensive method of quantifying drug effects on malignant cells. This makes it a potentially useful method for screening new anticancer drugs in human cancer cell lines.     

The Capacity of Red Blood Cells to Reduce Nitrite Determines Nitric Oxide Generation under Hypoxic Conditions

Nitric oxide (NO) is a key regulator of vascular tone. Endothelial nitric oxide synthase (eNOS) is responsible for NO generation under normoxic conditions. Under hypoxia however, eNOS is inactive and red blood cells (RBC) provide an alternative NO generation pathway from nitrite to regulate hypoxic vasodilation. While nitrite reductase activity of hemoglobin is well acknowledged, little is known about generation of NO by intact RBC with physiological hemoglobin concentrations. We aimed to develop and apply a new approach to provide insights in the ability of RBC to convert nitrite into NO under hypoxic conditions. We established a novel experimental setup to evaluate nitrite uptake and the release of NO from RBC into the gas-phase under different conditions. NO measurements were similar to well-established clinical measurements of exhaled NO. Nitrite uptake was rapid, and after an initial lag phase NO release from RBC was constant in time under hypoxic conditions. The presence of oxygen greatly reduced NO release, whereas inhibition of eNOS and xanthine oxidoreductase (XOR) did not affect NO release. A decreased pH increased NO release under hypoxic conditions. Hypothermia lowered NO release, while hyperthermia increased NO release. Whereas fetal hemoglobin did not alter NO release compared to adult hemoglobin, sickle RBC showed an increased ability to release NO. Under all conditions nitrite uptake by RBC was similar. This study shows that nitrite uptake into RBC is rapid and release of NO into the gas-phase continues for prolonged periods of time under hypoxic conditions. Changes in the RBC environment such as pH, temperature or hemoglobin type, affect NO release.     

Variations in the Adenylate Energy Charge During Phased Growth (Cell Cycle) of Candida utilis Under Energy Excess and Energy-Limiting Growth Conditions

The variations in the levels of adenine nucleotides during the phased growth (cell cycle) of the yeast Candida utilis growing under nitrogen, sulfate, or iron limitation with glycerol as carbon source have been determined. Synchronous cultures were obtained by the continuous phasing technique, and the results were compared with those of chemostat cultures growing at similar growth rates and under the same types of nutrient limitation. Whereas the chemostat experiments indicated only the average energy status of cultures growing at random, results from phased cultures showed that the adenylate energy charge, defined as (ATP + (1/2)ADP)/(ATP + ADP + AMP) (where ATP, ADP, and AMP signify adenosine 5'-triphosphate, -diphosphate, and -monophosphate, respectively), varied during the phased growth of the yeast. These variations were related to the stage of development of the cells and to the type of nutrient limitation. In every case the energy charge dropped to a low value during the first half of the phasing cycle (cell cycle). Whereas the energy charge was maintained at relatively high levels (ranging from 0.78 to 0.94), for sulfate- or nitrogen-limited cultures, it was very low when iron was the growth-limiting nutrient (0.44 to 0.78). In spite of the low energy charge, the yeast continued to grow under iron limitation. The main component of the adenylate pool of the iron-limited culture was ADP and not ATP as observed with other types of nutrient limitation. It is concluded that under iron limitation the growth of the organism is limited by energy and that under energy-limited growth the energy charge of a growing organism is maintained at low levels. The reason for maintaining a low energy charge in an energy-limited culture is discussed.     

Prostate carcinoma cells selected by long-term exposure to reduced oxygen tension show remarkable biochemical plasticity via modulation of superoxide, HIF-1alpha levels, and energy metabolism

Cancer cells are able to tolerate levels of O(2) that are damaging or lethal to normal cells; we hypothesize that this tolerance is the result of biochemical plasticity which maintains cellular homeostasis of both energy levels and oxidation state. In order to examine this hypothesis, we used different O(2) levels as a selective agent during long-term culture of DU145 prostate cancer cells to develop three isogenic cell lines that grow in normoxic (4%), hyperoxic (21%), or hypoxic (1%) O(2) conditions. Growth characteristics and O(2) consumption differed significantly between these cell lines without changes in ATP levels or altered sensitivity to 2-deoxy-D-glucose, an inhibitor of glycolysis. O(2) consumption was significantly higher in the hyperoxic line as was the level of endogenous superoxide. The hypoxic cell line regulated the chemical gradient of the proton motive force (PMF) independent of the electrical component without O(2)-dependent changes in Hif-1alpha levels. In contrast, the normoxic line regulated Hif-1alpha without tight regulation of the chemical component of the PMF noted in the hypoxic cell line. From these studies, we conclude that selection of prostate cancer cells by long-term exposure to low ambient levels of O(2) resulted in cells with unique biochemical properties in which energy metabolism, reactive oxygen species (ROS), and HIF-1alpha levels are modulated to allow cell survival and growth. Thus, cancer cells exhibit remarkable biochemical plasticity in response to various O(2) levels.     

NADH-dependent metabolism of nitric oxide in alfalfa root cultures expressing barley hemoglobin

Transgenic alfalfa (Medicago sativa L.) root cultures expressing sense and antisense barley (Hordeum vulgare L.) hemoglobin were examined for their ability to metabolize NO. Extracts from lines overexpressing hemoglobin had approximately twice the NO conversion rate of either control or antisense lines under normoxic conditions. Only the control line showed a significant increase in the rate of NO degradation when placed under anaerobic conditions. The decline in NO was dependent on the presence of reduced pyridine nucleotide, with the NADH-dependent rate being about 2.5 times faster than the NADPH-dependent rate. Most of the activity was found in the cytosolic fraction of the extracts, while only small amounts were found in the cell wall, mitochondria, and 105,000-g membrane fraction. The NADH-dependent NO conversion exhibited a broad pH optimum in the range 7–8 and a strong affinity to NADH and NADPH (K _m 3 M for both). It was sensitive to diphenylene iodonium, an inhibitor of flavoproteins. The activity was strongly reduced by applying antibodies raised against recombinant barley hemoglobin. Extracts of Escherichia coli overexpressing barley hemoglobin showed a 4-fold higher rate of NO metabolism as compared to non-transformed cells. The NADH/NAD and NADPH/NADP ratios were higher in lines underexpressing hemoglobin, indicating that the presence of hemoglobin has an effect on these ratios. They were increased under hypoxia and antimycin A treatment. Alfalfa root extracts exhibited methemoglobin reductase activity, using either cytochrome c or recombinant barley hemoglobin as substrates. There was a correspondence between NO degradation and nitrate formation. The activity was eluted from a Superose 12 column as a single peak with molecular weight of 35±4 kDa, which corresponds to the size of the hemoglobin dimer. The results are consistent with an NO dioxygenase-like activity, with hemoglobin acting in concert with a flavoprotein, to metabolize NO to nitrate utilizing NADH as the electron donor.Abbreviation Hb Hemoglobin     

Relationship between culture conditions and the dependency on mitochondrial function of mammalian cell proliferation.

In cultured mammalian cells, the relationship was investigated between mitochondrial function and proliferation under various culture conditions. Continuous inhibition of the expression of the mitochondrial genome was used to reduce the activity of enzymes involved in oxidative phosphorylation by 50% at every cell division. Under these conditions, culturing in relatively poor media resulted in arrest of the proliferation of most cell lines after 1 cell division. This was preceded by decreasing levels of ATP and increasing levels of ADP, suggesting that the ATP-generating capacity of the cells was limiting. Culturing in richer media led to arrest of the proliferation after 5 to 6 divisions, but accumulation of ADP was not observed. Addition of pyruvate to rich culture media and, at least for 1 cell line, increasing the CO2 levels, completely prevented proliferation arrest. Inability to synthesise metabolic precursors via mitochondrial intermediary metabolism probably explains growth arrest of cells cultured in rich media. Pyruvate and CO2 were, however, without effect on the proliferation arrest of cells cultured in relatively poor media. Therefore, pyruvate dependency for growth of cells without functional mitochondria holds true only under culture conditions where the ATP-generating capacity of the cells is not limiting.     

Nitric oxide induces monosaccharide accumulation through enzyme S‐nitrosylation

Nitric oxide (NO) is extensively involved in various growth processes and stress responses in plants; however, the regulatory mechanism of NO‐modulated cellular sugar metabolism is still largely unknown. Here, we report that NO significantly inhibited monosaccharide catabolism by modulating sugar metabolic enzymes through S‐nitrosylation (mainly by oxidizing dihydrolipoamide, a cofactor of pyruvate dehydrogenase). These S‐nitrosylation modifications led to a decrease in cellular glycolysis enzymes and ATP synthase activities as well as declines in the content of acetyl coenzyme A, ATP, ADP‐glucose and UDP‐glucose, which eventually caused polysaccharide‐biosynthesis inhibition and monosaccharide accumulation. Plant developmental defects that were caused by high levels of NO included delayed flowering time, retarded root growth and reduced starch granule formation. These phenotypic defects could be mediated by sucrose supplementation, suggesting an essential role of NO‐sugar cross‐talks in plant growth and development. Our findings suggest that molecular manipulations could be used to improve fruit and vegetable sweetness.     

Biochemical characterization of human S-nitrosohemoglobin. Effects on oxygen binding and transnitrosation.

S-Nitrosation of cysteine beta93 in hemoglobin (S-nitrosohemoglobin (SNO-Hb)) occurs in vivo, and transnitrosation reactions of deoxygenated SNO-Hb are proposed as a mechanism leading to release of NO and control of blood flow. However, little is known of the oxygen binding properties of SNO-Hb or the effects of oxygen on transnitrosation between SNO-Hb and the dominant low molecular weight thiol in the red blood cell, GSH. These data are important as they would provide a biochemical framework to assess the physiological function of SNO-Hb. Our results demonstrate that SNO-Hb has a higher affinity for oxygen than native Hb. This implies that NO transfer from SNO-Hb in vivo would be limited to regions of extremely low oxygen tension if this were to occur from deoxygenated SNO-Hb. Furthermore, the kinetics of the transnitrosation reactions between GSH and SNO-Hb are relatively slow, making transfer of NO+ from SNO-Hb to GSH less likely as a mechanism to elicit vessel relaxation under conditions of low oxygen tension and over the circulatory lifetime of a given red blood cell. These data suggest that the reported oxygen-dependent promotion of S-nitrosation from SNO-Hb involves biochemical mechanisms that are not intrinsic to the Hb molecule.     

H Efflux and Hexose Transport under Imposed Energy Status in Maize Root Tips

The relationship between changes in H⁺ flux and sugar transport in maize Zea mays L. DEA root tips have been investigated using two methods for controlling the cellular nucleotide level: (a) incubation in the presence of a glucose analog, the 2-deoxyglucose, which decreased the ATP level to less than 15% of its initial value within 60 minutes without changing the ADP and AMP levels; (b) an hypoxic treatment which also decreased the ATP level but with a concomitant rise in ADP and AMP. In both cases the rate of hexose transport was not modified until ATP had dropped to 70% of its initial value; then it decreased with the cellular ATP level. The residual uptake rate at very low ATP concentrations still represented 50% of the maximum rate with the dGlc treatment but only the diffusion rate in anoxia. H⁺ efflux was abolished in anoxia but not by the 2-deoxyglucose treatment, in spite of a lower cellular ATP concentration. Our results are consistent with an inhibition of H⁺-ATPase activity in anoxia by the high levels of cellular ADP and AMP, and provide in vivo evidence that sugar uptake is dependent upon the proton motive force rather than cellular ATP concentration. The absence of stimulation of H⁺ extrusion by ferricyanide in either normoxic or hypoxic conditions suggests that a redox system does not appear to contribute to H⁺ secretion under the conditions of this investigation.     

Heterologous complementation of the Klaac null mutation of Kluyveromyces lactis by the Saccharomyces cerevisiae AAC3 gene encoding the ADP/ATP carrier

The KlAAC gene, encoding the ADP/ATP carrier, has been assumed to be a single gene in Kluyveromyces lactis, an aerobic, petite-negative yeast species. The Klaac null mutation, which causes a respiratory-deficient phenotype, was fully complemented by AAC2, the Saccharomyces cerevisiae major gene for the ADP/ATP carrier and also by AAC1, a gene that is poorly expressed in S. cerevisiae. In this study, we demonstrate that the Klaac null mutation is partially complemented by the ScAAC3 gene, encoding the hypoxic ADP/ATP carrier isoform, whose expression in S. cerevisiae is prevented by oxygen. Once introduced into K. lactis, the AAC3 gene was expressed both under aerobic and under partial anaerobic conditions but did not support the growth of K. lactis under strict anaerobic conditions.     

Deciphering the physiological response of Escherichia coli under high ATP demand

One long‐standing question in microbiology is how microbes buffer perturbations in energy metabolism. In this study, we systematically analyzed the impact of different levels of ATP demand in Escherichia coli under various conditions (aerobic and anaerobic, with and without cell growth). One key finding is that, under all conditions tested, the glucose uptake increases with rising ATP demand, but only to a critical level beyond which it drops markedly, even below wild‐type levels. Focusing on anaerobic growth and using metabolomics and proteomics data in combination with a kinetic model, we show that this biphasic behavior is induced by the dual dependency of the phosphofructokinase on ATP (substrate) and ADP (allosteric activator). This mechanism buffers increased ATP demands by a higher glycolytic flux but, as shown herein, it collapses under very low ATP concentrations. Model analysis also revealed two major rate‐controlling steps in the glycolysis under high ATP demand, which could be confirmed experimentally. Our results provide new insights on fundamental mechanisms of bacterial energy metabolism and guide the rational engineering of highly productive cell factories.     

Metabolic profiling of hypoxic cells revealed a catabolic signature required for cell survival

Hypoxia is one of the features of poorly vascularised areas of solid tumours but cancer cells can survive in these areas despite the low oxygen tension. The adaptation to hypoxia requires both biochemical and genetic responses that culminate in a metabolic rearrangement to counter-balance the decrease in energy supply from mitochondrial respiration. The understanding of metabolic adaptations under hypoxia could reveal novel pathways that, if targeted, would lead to specific death of hypoxic regions. In this study, we developed biochemical and metabolomic analyses to assess the effects of hypoxia on cellular metabolism of HCT116 cancer cell line. We utilized an oxygen fluorescent probe in anaerobic cuvettes to study oxygen consumption rates under hypoxic conditions without the need to re-oxygenate the cells and demonstrated that hypoxic cells can maintain active, though diminished, oxidative phosphorylation even at 1% oxygen. These results were further supported by in situ microscopy analysis of mitochondrial NADH oxidation under hypoxia. We then used metabolomic methodologies, utilizing liquid chromatography-mass spectrometry (LC-MS), to determine the metabolic profile of hypoxic cells. This approach revealed the importance of synchronized and regulated catabolism as a mechanism of adaptation to bioenergetic stress. We then confirmed the presence of autophagy under hypoxic conditions and demonstrated that the inhibition of this catabolic process dramatically reduced the ATP levels in hypoxic cells and stimulated hypoxia-induced cell death. These results suggest that under hypoxia, autophagy is required to support ATP production, in addition to glycolysis, and that the inhibition of autophagy might be used to selectively target hypoxic regions of tumours, the most notoriously resistant areas of solid tumours.     

Intrinsic properties of muscle satellite cells are changed in response to long-term selection of mice for different growth traits

Satellite cell cultures were derived from mice selected long-term over 70 generations for body weight (DU-6, growth), carcass protein amount (DU-6P, protein) and an index combining body weight and endurance treadmill performance (DU-6+LB, growth + fitness) at 42 days of age and from an unselected control line (DU-Ks). They were grown under identical environmental conditions to examine intrinsic cellular differences in proliferation, protein metabolism and responsiveness to growth factors. Growth kinetics (DNA and protein amounts) were determined over a 12-day period. During exponential growth, all growth-selected cultures grew faster than the control culture: (DU-6+LB=DU-6P)>DU-6>DU-Ks. The differences in DNA and protein levels were maintained until day 8. DU-Ks cultures reached similar levels as the growth (DU-6) and protein (DU-6P) cultures in terms of DNA at day 12 of cultivation. Thus, the cultures from the growth and protein lines, but not from the growth + fitness line, exhibited larger protein:DNA ratios (cell size) than the control cultures. Cell cultures from the selected lines were more responsive to serum and epidermal growth factor in terms of [(3)H] thymidine incorporation into DNA, whereas no stimulation by insulin or insulin-like growth factor-I was detectable in cultures from selected lines or controls. During differentiation, protein metabolism in cultures from selected lines was characterised by higher rates of protein synthesis (PS) and degradation (PD), as measured by [(3)H] phenylalanine incorporation or release, respectively, than in control cells. The ratios of the relative differences from the control in PS and PD were only >1.0 in the growth and protein lines. In conclusion, long-term selection for growth therefore modifies the intrinsic capability of satellite cells for proliferation and protein metabolism, with changes being dependent on the selection trait.     

Quantitative phosphoproteomic analysis of prion-infected neuronal cells

Following cultivation of distinct mesenchymal stem cell (MSC) populations derived from human umbilical cord under hypoxic conditions (between 1.5% to 5% oxygen (O₂)) revealed a 2- to 3-fold reduced oxygen consumption rate as compared to the same cultures at normoxic oxygen levels (21% O₂). A simultaneous measurement of dissolved oxygen within the culture media from 4 different MSC donors ranged from 15 μmol/L at 1.5% O₂ to 196 μmol/L at normoxic 21% O₂. The proliferative capacity of the different hypoxic MSC populations was elevated as compared to the normoxic culture. This effect was paralleled by a significantly reduced cell damage or cell death under hypoxic conditions as evaluated by the cellular release of LDH whereby the measurement of caspase3/7 activity revealed little if any differences in apoptotic cell death between the various cultures. The MSC culture under hypoxic conditions was associated with the induction of hypoxia-inducing factor-alpha (HIF-1α) and an elevated expression of energy metabolism-associated genes including GLUT-1, LDH and PDK1. Concomitantly, a significantly enhanced glucose consumption and a corresponding lactate production could be observed in the hypoxic MSC cultures suggesting an altered metabolism of these human stem cells within the hypoxic environment.