Glucose Consumption Rate Research Articles

EngineeringCorynebacterium crenatumfor enhancing succinic acid production

Succinic acid is valuable due to its historical use in food industry. In this paper, Corynebacterium crenatum was engineered to enhance succinic acid production and the fermentation properties of engineered strains were investigated. The lactate dehydrogenase gene (ldhA) inactivation mutant was first constructed, and then the pyruvate carboxylase gene (pyc) or phosphoenolpyruvate carboxylase gene (ppc) was expressed in the ldhA mutant. The ldhA inactivation resulted in a 52% decrease on glucose consumption, and the succinic acid yield produced by the ldhA mutant was 0.62 g/g, representing a 2-fold increase compared with the yield obtained from the original strain. Overexpression of pyc gene could improve the glucose consumption rate, resulting in a 37% increase compared with the ldhA mutant strain. Meanwhile, the final succinic acid concentration represented a 50% increase than the ldhA mutant strain, and a 106% increase than the original strain. Overexpression of ppc gene had no significant effect. Practical applications The biobased succinic acid and the derivative can be used as food additives. C. crenatum is used for producing amino acid for many years, and it was found that succinic acid and lactic acid are the major products when fermented under anaerobic conditions. This study suggests that C. crenatum can be utilized as a potential cell factory for producing succinic acid after genetic modification. The results obtained in this paper could contribute to the depth development of C. crenatum for producing succinic acid or other products in food industry, not only for amino acid production.

Development of a shake tube-based scale-down model for perfusion cultures.

The use of benchtop bioreactors (BRs) for the development of mammalian cell perfusion cultures is expensive and time consuming, given its complexity in equipment and operation. Scale-down models, going from liter to milliliter scale, are needed to support the rapid determination of suitable operating conditions in terms of viable cell density (VCD), perfusion rate, and medium composition. In this study, we compare the performance of steady-state perfusion cultures in orbitally shaken tube and BR systems for a given Chinese hamster ovary cell line. The developed scale-down model relied on a daily workflow designed to keep the VCD constant at specific target values. This includes: cell count, removal of excessive cells (bleeding), spin down of remaining cells, harvest of cell-free supernatant, and resuspension in fresh medium. Steady-state cultures at different VCD values, medium exchange rates and working volumes were evaluated. Shake-tube perfusion cultures allowed the prediction of cell-specific growth, glucose consumption, ammonia, and monoclonal antibody production rates for much larger BRs, but not lactate (LAC) production rates. Although charge variant profiles remained comparable, different glycosylation patterns were obtained. The differences in LAC production and glycosylation probably resulted from the discontinuous medium exchange, the poor carbon dioxide removal, and the deficient pH control. Therefore, if requested by the specific process to be developed, product quality has to be fine-tuned directly in the BR system. Altogether, the developed strategy provides a useful scale-down model for the design and optimization of perfusion cultures with strong savings in time and media consumption.

The Long Noncoding RNA Lncenc1 Maintains Naive States of Mouse ESCs by Promoting the Glycolysis Pathway.

SummaryThe naive embryonic stem cells (nESCs) display unique characteristics compared with the primed counterparts, but the underlying molecular mechanisms remain elusive. Here we investigate the functional roles of Lncenc1, a highly abundant long noncoding RNA in nESCs. Knockdown or knockout of Lncenc1 in mouse nESCs leads to a significantly decreased expression of core pluripotency genes and a significant reduction of colony formation capability. Furthermore, upon the depletion of Lncenc1, the expression of glycolysis-associated genes is significantly reduced, and the glycolytic activity is substantially impaired, as indicated by a more than 50% reduction in levels of glucose consumption, lactate production, and extracellular acidification rate. Mechanistically, Lncenc1 interacts with PTBP1 and HNRNPK, which regulate the transcription of glycolytic genes, thereby maintaining the self-renewal of nESCs. Our results demonstrate the functions of Lncenc1 in linking energy metabolism and naive state of ESCs, which may enhance our understanding of the molecular basis underlying naive pluripotency.

Physiological Response of Corynebacterium glutamicum to Increasingly Nutrient-Rich Growth Conditions.

To ensure economic competitiveness, bioprocesses should achieve maximum productivities enabled by high growth rates (μ) and equally high substrate consumption rates (qS) as a prerequisite of sufficient carbon-to-product conversion. Both traits were investigated and improved via bioprocess engineering approaches studying the industrial work horse Corynebacterium glutamicum. Standard minimal medium CGXII with glucose as sole carbon source was supplemented with complex brain-heart-infusion (BHI) or amino acid (AA) cocktails. Maximum μ of 0.67 h-1 was exclusively observed in 37 g BHI L-1 whereas only minor growth stimulation was found after AA supplementation (μ = 0.468 h-1). Increasing glucose consumption rates (qGlc) were solely observed in certain dosages of BHI (1–10 g L-1), while 37 g BHI L-1 and AA addition revealed qGlc below the reference experiments. Moreover, BHI supplementation revealed Monod-type saturation kinetics of μ (KBHI = 2.73 g BHI L-1) referring to the preference of non-AAs as key boosting nutrients. ATP-demands under reference, 1 g BHI L-1, and AA conditions were nearly constant but halved in BHI concentrations above 5 g L-1 reflecting the energetic advantage of consuming complex nutrient components in addition to “simple” building blocks such as AAs. Furthermore, C. glutamicum revealed maximum biomass per carbon yields of about 18 gCDW C-mol-1 irrespective of the medium. In AA supplementation experiments, simultaneous uptake of 17 AAs was observed, maximum individual consumption rates determined, and L-asparagine and L-glutamine were distinguished as compounds with the highest consumption rates. Employment of the expanded stoichiometric model iMG481 successfully reproduced experimental results and revealed the importance of C. glutamicum’s transaminase network to compensate needs of limiting AA supply. Model-based sensitivity studies attributed the highest impact on μ to AAs with high ATP and NADPH demands such as L-tryptophan or L-phenylalanine.

Bioethanol production in vacuum membrane distillation bioreactor by permeate fractional condensation and mechanical vapor compression with polytetrafluoroethylene (PTFE) membrane

A vacuum membrane distillation bioreactor (VMDBR) by permeate fractional condensation and mechanical vapor compression with PTFE membrane was developed for bioethanol production. Cell concentration of 11.5 g/L, glucose consumption rate of 5.2 g/L/h and ethanol productivity of 2.3 g/L/h could be obtained with fermentation continues lasting for 140 h. Membrane flux of over 10 kg/m2/h could be obtained for model solution separation. Higher temperature and flow rate could promote membrane separation. Membrane flux could be reduced to about 2000 g/m2/h with fermentation proceeding owing to the deposited cell on membrane surface. The membrane separation performance could be resumed by water rinse. High ethanol concentration of 421 g/L could be obtained by permeate fractional condensation with the process separation factor increased to 19.2. Energy of only 14 MJ/kg was required in VMDBR and the energy consumption would be reduced further if the compressed vapor could be used to heat the feed.

High glucose and low specific cell growth but not mild hypothermia improve specific r-protein productivity in chemostat culture of CHO cells.

In the biopharmaceutical sector, Chinese hamster ovary (CHO) cells have become the host of choice to produce recombinant proteins (r-proteins) due to their capacity for correct protein folding, assembly, and posttranslational modification. However, the production of therapeutic r-proteins in CHO cells is expensive and presents insufficient production yields for certain proteins. Effective culture strategies to increase productivity (qp) include a high glucose concentration in the medium and mild hypothermia (28–34 °C), but these changes lead to a reduced specific growth rate. To study the individual and combined impacts of glucose concentration, specific growth rate and mild hypothermia on culture performance and cell metabolism, we analyzed chemostat cultures of recombinant human tissue plasminogen activator (rh-tPA)-producing CHO cell lines fed with three glucose concentrations in feeding media (20, 30 and 40 mM), at two dilution rates (0.01 and 0.018 1/h) and two temperatures (33 and 37 °C). The results indicated significant changes in cell growth, cell cycle distribution, metabolism, and rh-tPA productivity in response to the varying environmental culture conditions. High glucose feed led to constrained cell growth, increased specific rh-tPA productivity and a higher number of cells in the G2/M phase. Low specific growth rate and temperature (33 °C) reduced glucose consumption and lactate production rates. Our findings indicated that a reduced specific growth rate coupled with high feed glucose significantly improves r-protein productivity in CHO cells. We also observed that low temperature significantly reduced qp, but not cell growth when dilution rate was manipulated, regardless of the glucose concentration or dilution rate. In contrast, we determined that feed glucose concentration and consumption rate were the dominant aspects of the growth and productivity in CHO cells by using multivariate analysis.

Inferring oncoenzymes in a genome-scale metabolic network for hepatocytes using bilevel optimization framework

Abstract Cancer cells exhibit unusual metabolic activity, characterized by high rates of glucose consumption and lactate production, even under aerobic conditions, as well as increased glutamine catabolism and amino acid metabolism. Based on the behaviors of metabolic reprogramming, we can infer oncogenesis from a genome-scale model of cancer cell metabolism. This study establishes a bilevel optimization formulation that integrates the genome-scale metabolic model of hepatocytes, the Warburg hypothesis, and LC/MS experimental data to detect multiple-hit enzyme deficiencies that induce metabolic reprogramming in hepatocytes. A nested hybrid differential evolution algorithm was employed to solve the bilevel optimization problem. The results predicted dopa decarboxylase (DDC) to be an influential enzyme that causes oncogenesis. A cluster analysis of the flux variations for different enzyme deficiencies obtained through a flux variability analysis showed that DDC is a dominant overexpressed enzyme, and it was classified into a group with similar trends of flux and metabolite alternations.

Calcium and Magnesium Urinary Excretion in Dairy Cows with Different Fee of Glucose Metabolization

Background: The post-partum period in dairy cows is accompanied by a low glucose metabolism in adipose tissue and skeletal muscle tissue, being glucose conducted to the milk production. In humans, low glucose metabolism is associated with metabolic syndromes, the high glucose levels reduce tubular reabsorption of Magnesium (Mg) and Calcium (Ca), leading to hypomagnesemia and hypocalcemia. These minerals are important to the dairy cow, as their decrease leads to diseases. The aim of this study was to evaluate the relationship between glucose metabolism rate with the urinary excretion of Ca and Mg in multiparous dairy cows during the post-partum period.Materials, Methods & Results: Twenty dairy cows were used from a commercial farm southern Brazil, in the semi-extensive system. Glucose tolerance tests were performed (TTG) on day 9 relative to calving. The cows were categorized into three groups according to the glucose metabolism rate (area under the glucose curve, glucose half-life and glucose consumption rate): High Glucose Metabolization (GA); Intermediate Glucose Metabolizing (GI); and Low Glucose Metabolization (GL). Blood and urine samples were collected on days 0, + 3, + 6, + 9, +16 and +2 3 in relation to calving for to determine the levels of Ca, Mg, insulin (Ins), non-esterified fatty acids (NEFA) and Glu. In urine was evaluated the excretion of Ca and Mg. The cows were milked twice a day (at 3:00 a.m. and 3:00 p.m.) and the milk yield (kg/cow) was recorded daily and averages were generated every five days from day 15 to day 60 postpartum. The statistical analyses were performed with the MIXED procedure to assess the main effect of group, time (in days) and their interaction by using version 9.2 SAS software. The influence of the different rates of glucose metabolism on milk production was observed, the GB group had a production than GH group (30.88 ± 1.44 kg vs 23.96 ± 1.43 kg, P < 0.01), but did not differ from GI. The GL group showed higher levels of Glu compared to GA (P < 0.05). The plasma Ca levels were higher in GL (P < 0.05) compared with GH. The NEFA, insulin, and excretion of minerals did not differ between groups (P > 0.05).Discussion: The low glucose metabolism in humans causes an increase in the excretion of Ca and Mg urine, however, in the animals studied, these changes were not observed. This result can be attributed to the fact that insulin resistance is transitory in dairy cattle. The higher glucose levels in the GL group are related due to the lower capacity of glucose entry in the peripheral tissues (adipose and skeletal muscle), which reflected in the higher milk production observed this group. However, the higher calcium concentrations were not expected, since the release of insulin by β-pancreatic cells is dependent on calcium. Possibly, these higher calcium levels in GB, are related to higher milk production, requiring a greater amount of calcium for the production of casein, increasing bone mobilization, intestinal absorption. The energy metabolites, non-esterified fatty acids and insulin, did not differ between groups, suggesting that the animals did not present different metabolic conditions. We conclude that multiparous dairy cows with low glucose metabolism rate (GB) have higher levels of glucose after delivery and increased milk production. The metabolism rate of glucose did not influence the excretion of the Ca and Mg minerals.

A precision therapeutic strategy for hexokinase 1-null, hexokinase 2-positive cancers

BackgroundPrecision medicine therapies require identification of unique molecular cancer characteristics. Hexokinase (HK) activity has been proposed as a therapeutic target; however, different hexokinase isoforms have not been well characterized as alternative targets. While HK2 is highly expressed in the majority of cancers, cancer subtypes with differential HK1 and HK2 expression have not been characterized for their sensitivities to HK2 silencing.MethodsHK1 and HK2 expression in the Cancer Cell Line Encyclopedia dataset was analyzed. A doxycycline-inducible shRNA silencing system was used to examine the effect of HK2 knockdown in cultured cells and in xenograft models of HK1−HK2+ and HK1+HK2+ cancers. Glucose consumption and lactate production rates were measured to monitor HK activity in cell culture, and 18F-FDG PET/CT was used to monitor HK activity in xenograft tumors. A high-throughput screen was performed to search for synthetically lethal compounds in combination with HK2 inhibition in HK1−HK2+ liver cancer cells, and a combination therapy for liver cancers with this phenotype was developed. A metabolomic analysis was performed to examine changes in cellular energy levels and key metabolites in HK1−HK2+ cells treated with this combination therapy. The CRISPR Cas9 method was used to establish isogenic HK1+HK2+ and HK1−HK2+ cell lines to evaluate HK1−HK2+ cancer cell sensitivity to the combination therapy.ResultsMost tumors express both HK1 and HK2, and subsets of cancers from a wide variety of tissues of origin express only HK2. Unlike HK1+HK2+ cancers, HK1−HK2+ cancers are sensitive to HK2 silencing-induced cytostasis. Synthetic lethality was achieved in HK1−HK2+ liver cancer cells, by the combination of DPI, a mitochondrial complex I inhibitor, and HK2 inhibition, in HK1−HK2+ liver cancer cells. Perhexiline, a fatty acid oxidation inhibitor, further sensitizes HK1−HK2+ liver cancer cells to the complex I/HK2-targeted therapeutic combination. Although HK1+HK2+ lung cancer H460 cells are resistant to this therapeutic combination, isogenic HK1KOHK2+ cells are sensitive to this therapy.ConclusionsThe HK1−HK2+ cancer subsets exist among a wide variety of cancer types. Selective inhibition of the HK1−HK2+ cancer cell-specific energy production pathways (HK2-driven glycolysis, oxidative phosphorylation and fatty acid oxidation), due to the unique presence of only the HK2 isoform, appears promising to treat HK1−HK2+ cancers. This therapeutic strategy will likely be tolerated by most normal tissues, where only HK1 is expressed.

Overexpression of THI4 and HAP4 Improves Glucose Metabolism and Ethanol Production in Saccharomyces cerevisiae.

Redox homeostasis is essential to the maintenance of cell metabolism. Changes in the redox state cause global metabolic and transcriptional changes. Our previous study indicated that the overexpression of NADH oxidase in Saccharomyces cerevisiae led to increased glucose consumption and ethanol production. Gene expression related to thiamine synthesis and osmotolerance as well as HAP4 expression was increased in response to redox change caused by the overexpression of NADH oxidase. To identify detailed relationships among cofactor levels, thiamine synthesis, expression of HAP4, and osmotolerance, and to determine whether these changes are interdependent, THI4 and HAP4 were overexpressed in S. cerevisiae BY4741. The glucose consumption rate of THI4-overexpressing strain (thi4-OE) was the highest, followed by HAP4-overexpressing strain (hap4-OE) > NADH oxidase-overexpressing strain (nox-OE) > control strain (con), while strain hap4-OE showed the highest concentration of ethanol after 26 h of fermentation. Reduced glycerol production and increased osmotolerance were observed in thi4-OE and hap4-OE, as well as in nox-OE. HAP4 globally regulated thiamine synthesis, biomass synthesis, respiration, and osmotolerance of cells, which conferred the recombinant strain hap4-OE with faster glucose metabolism and enhanced stress resistance. Moreover, overexpression of HAP4 might extend the life span of cells under caloric restriction by lowering the NADH level. Although overexpression of THI4 and HAP4 induced various similar changes at both the metabolic and the transcriptional level, the regulatory effect of THI4 was more limited than that of HAP4, and was restricted to the growth phase of cells. Our findings are expected to benefit the bio-ethanol industry.

Regulation of Cellular Metabolism by High-Risk Human Papillomaviruses

The alteration of glucose metabolism is one of the first biochemical characteristics associated with cancer cells since most of these cells increase glucose consumption and glycolytic rates even in the presence of oxygen, which has been called “aerobic glycolysis” or the Warburg effect. Human papillomavirus (HPV) is associated with approximately 5% of all human cancers worldwide, principally to cervical cancer. E6 and E7 are the main viral oncoproteins which are required to preserve the malignant phenotype. These viral proteins regulate the cell cycle through their interaction with tumor suppressor proteins p53 and pRB, respectively. Together with the viral proteins E5 and E2, E6 and E7 can favor the Warburg effect and contribute to radio- and chemoresistance through the increase in the activity of glycolytic enzymes, as well as the inhibition of the Krebs cycle and the respiratory chain. These processes lead to a fast production of ATP obtained by Warburg, which could help satisfy the high energy demands of cancer cells during proliferation. In this way HPV proteins could promote cancer hallmarks. However, it is also possible that during an early HPV infection, the Warburg effect could help in the achievement of an efficient viral replication.

Enhancement of hydrogen production rate by high biomass concentrations of Thermotoga neapolitana

The objective of this study was to enhance the hydrogen production rate of dark fermentation in batch operation. For the first time, the hyperthermophilic pure culture of Thermotoga neapolitana cf. Capnolactica was applied at elevated biomass concentrations. The increase of the initial biomass concentration from 0.46 to 1.74 g cell dry weight/L led to a general acceleration of the fermentation process, reducing the fermentation time of 5 g glucose/L down to 3 h with a lag phase of 0.4 h. The volumetric hydrogen production rate increased from 323 (±11) to 654 (±30) mL/L/h with a concomitant enhancement of the biomass growth and glucose consumption rate. The hydrogen yield of 2.45 (±0.09) mol H2/mol glucose, the hydrogen concentration of 68% in the produced gas and the composition of the end products in the digestate, i.e. 62.3 (±2.5)% acetic acid, 23.5 (±2.9)% lactic acid and 2.3 (±0.1)% alanine, remained unaffected at increasing biomass concentrations.

Synthesis of a thin-layer gelatin nanofiber mat for cultivating retinal cell

Thin-layer gelatin nanofiber mats were fabricated as a biodegradable scaffold for proliferating human retinal pigment epithelium. Together with MTT assay, the glucose consumption rate, lactate formation, and lactate dehydrogenase activity of the human retinal pigment epithelium cells—on the gelatin nanofibers—were analyzed as indicators for cell growth and viability. The results showed that gelatin nanofiber did not make any toxic effect on the cells and the growth rate was comparable to the tissue culture plates. Using the fabricated thin-layer nanofibers let the by-product to leave which in turn cause less adverse effect on the cells. The biodegradability and stability of the gelatin nanofibers were optimized as a function of reaction time.

Effects Of Oxygen Concentration And Culture Time On Porcine Nucleus Pulposus Cell Metabolism: An In Vitro Study

Low back pain is a common ailment that affects millions of individuals each year and is linked to degeneration of the intervertebral discs in the spine. Intervertebral disc degeneration is known to result from an imbalance in anabolic and catabolic activity by disc cells. Due to the avascular nature of the intervertebral disc, oxygen deficiency may occur in the central nucleus pulposus (NP). The resulting hypoxia affects matrix regulation and energy metabolism of disc cells, although the mechanisms are not fully understood. This study investigates in vitro glucose consumption and gene expression by NP cells over time under varying oxygen tensions. Notochordal porcine NP cells were cultured in agarose discs at 21, 5, or 1% oxygen tension for 1, 5, or 10 days. The expression of 10 key matrix genes, as well as Brachyury (T), by NP cells was analyzed using RT-PCR. Glucose consumption was measured using a two-point method. Results show that culture time and oxygen tension significantly affect glucose consumption rates by porcine NP cells. There were also significant changes in T expression based on oxygen level and culture time. The 1% oxygen tension had a significantly higher T expression on day 10 than the other two groups, which may indicate a better maintenance of the notochordal phenotype. MMP 1 and 13 expression increased over time for all groups, while only the 5% group showed an increase over time for MMP 3. TIMP expression followed the direction of MMPs but to a lesser magnitude. Five percent and twenty-one percent oxygen tensions led to decreases in anabolic gene expression while 1% led to increases. Oxygen concentration and culture time significantly impacted glucose consumption rate and the gene expression of matrix regulatory genes with hypoxic conditions most accurately maintaining the proper NP phenotype. This information is valuable not only for understanding disc pathophysiology, but also for harnessing the potential of notochordal NP cells in therapeutic applications.

Cell size and morphological properties of yeast Saccharomyces cerevisiae in relation to growth temperature.

Cell volume is an important parameter for modelling cellular processes. Temperature-induced variability of cellular size, volume, intracellular granularity, a fraction of budding cells of yeast Saccharomyces cerevisiae CEN.PK 113-7D (in anaerobic glucose unlimited batch cultures) were measured by flow cytometry and matched with the performance of the biomass growth (maximal specific growth rate (μmax), specific rate of glucose consumption, the rate of maintenance, biomass yield on glucose). The critical diameter of single cells was 7.94 μm and it is invariant at growth temperatures above 18.5°C. Below 18.5°C, it exponentially increases up to 10.2 μm. The size of the bud linearly depends on μmax, and it is between 50% at 5°C and 90% at 31°C of the averaged single cell. The intracellular granularity (side scatter channel (SSC)-index) negatively depends on μmax. There are two temperature regions (5-31°C vs. 33-40°C) where the relationship between SSC-index and various cellular parameters differ significantly. In supraoptimal temperature range (33-40°C), cells are less granulated perhaps due to a higher rate of the maintenance. There is temperature dependent passage through the checkpoints in the cell cycle which influences the μmax. The results point to the existence of two different morphological states of yeasts in these different temperature regions.

ASAS-SSR Triennnial Reproduction Symposium: Looking Back and Moving Forward-How Reproductive Physiology has Evolved: Fetal origins of impaired muscle growth and metabolic dysfunction: Lessons from the heat-stressed pregnant ewe.

Intrauterine growth restriction (IUGR) is the second leading cause of perinatal mortality and predisposes offspring to metabolic disorders at all stages of life. Muscle-centric fetal adaptations reduce growth and yield metabolic parsimony, beneficial for IUGR fetal survival but detrimental to metabolic health after birth. Epidemiological studies have reported that IUGR-born children experience greater prevalence of insulin resistance and obesity, which progresses to diabetes, hypertension, and other metabolic disorders in adulthood that reduce quality of life. Similar adaptive programming in livestock results in decreased birth weights, reduced and inefficient growth, decreased carcass merit, and substantially greater mortality rates prior to maturation. High rates of glucose consumption and metabolic plasticity make skeletal muscle a primary target for nutrient-sparing adaptations in the IUGR fetus, but at the cost of its contribution to proper glucose homeostasis after birth. Identifying the mechanisms underlying IUGR pathophysiology is a fundamental step in developing treatments and interventions to improve outcomes in IUGR-born humans and livestock. In this review, we outline the current knowledge regarding the adaptive restriction of muscle growth and alteration of glucose metabolism that develops in response to progressively exacerbating intrauterine conditions. In addition, we discuss the evidence implicating developmental changes in β adrenergic and inflammatory systems as key mechanisms for dysregulation of these processes. Lastly, we highlight the utility and importance of sheep models in developing this knowledge.

Use of glucose biosensors to measure extracellular glucose exudation by intertidal microphytobenthos in southern Tasmania.

Micro glucose biosensors were used to measure net extracellular glucose produced by natural microphytobenthos and three diatom cultures (Amphora coffeaeformis, Navicula menisculus, Nitzschia longissima) from southern Tasmania, Australia. They were exposed to a light gradient in either nutrient-replete or nutrient-limiting conditions. Glucose exudation in the natural communities increased with increased light but the response in the cultures was variable. Similarly, nutrient-replete conditions elicited lower rates of glucose exudation in the natural communities but produced variable species-specific responses in the cultures. Increased glucose exudation mostly correlated with a reduction in maximum quantum yield (Fv /Fm ). The same trend was observed in the natural communities for relative maximum electron transfer rates (rETRmax ) but responses in the cultures were again variable and species-specific. Responses of the three species to increased light and nutrient deficiency were variable, although glucose exudation, Fv /Fm and rETRmax was mostly lower in the nutrient-limited media. In a second set of experiments species/communities were treated with/without antibiotics. In the dark, glucose concentrations in treatments with antibiotics remained unchanged, while in those with bacteria, it fell rapidly. In the sediment communities, glucose consumption in the dark was ~25% the rate of exudation at the highest light level. In culture, exudation rates were up to 100% greater than those with active bacteria. Rates of glucose consumption in the dark in the antibiotic-treated samples were negligible and up to 104 times lower than those with active bacteria. These results demonstrate the important role extracellular glucose exudation has on maintaining an active microbial loop.

The histidine triad nucleotide-binding protein 2 (HINT-2) positively regulates hepatocellular energy metabolism.

The histidine triad nucleotide-binding protein 2 (HINT-2) is a mitochondrial adenosine phosphoramidase expressed in hepatocytes. The phenotype of Hint2 knockout ( Hint2-/-) mice includes progressive hepatic steatosis and lysine hyperacetylation of mitochondrial proteins, which are features of respiratory chain malfunctions. We postulated that the absence of HINT-2 induces a defect in mitochondria bioenergetics. Isolated Hint2-/- hepatocytes produced less ATP and generated a lower mitochondrial membrane potential than did Hint2+/+ hepatocytes. In extracellular flux analyses with glucose, the basal, ATP-linked, and maximum oxygen consumption rates (OCRs) were decreased in Hint2-/- hepatocytes and in HepG2 cells lacking HINT-2. Conversely, in HINT-2 overexpressing SNU-449 and HepG2 cells, the basal, ATP-linked, and maximum OCRs were increased. Similarly, with palmitate, basal and maximum OCRs were decreased in Hint2-/- hepatocytes, but they were increased in HINT-2 overexpressing HepG2 cells. When assayed with radiolabeled substrate, palmitate oxidation was reduced by 25% in Hint2-/- mitochondria. In respirometry assays, complex I- and II-driven, coupled and uncoupled respirations and complex IV KCN-sensitive respiration were reduced in Hint2-/- mitochondria. Furthermore, HINT-2 associated with cardiolipin and glucose-regulated protein 75 kDa. Our study shows decreased electron transfer and oxidative phosphorylation capacity in the absence of HINT-2. The bioenergetics deficit accumulated over time in hepatocytes lacking HINT-2 likely leads to the secondary outcome of steatosis.-Rajasekaran, R., Felser, A., Nuoffer, J.-M., Dufour, J.-F., St-Pierre, M. V. The histidine triad nucleotide-binding protein 2 (HINT-2) positively regulates hepatocellular energy metabolism.

Antimetabolic Effects of Polyphenols in Breast Cancer Cells: Focus on Glucose Uptake and Metabolism

In the last years, metabolic reprogramming became a new key hallmark of tumor cells. One of its components is a deviant energetic metabolism, known as Warburg effect—an aerobic lactatogenesis—characterized by elevated rates of glucose uptake and consumption with high-lactate production even in the presence of oxygen. Because many cancer cells display a greater sensitivity to glucose deprivation-induced cytotoxicity than normal cells, inhibitors of glucose cellular uptake (facilitative glucose transporter 1 inhibitors) and oxidative metabolism (glycolysis inhibitors) are potential therapeutic targets in cancer treatment. Polyphenols, abundantly contained in fruits and vegetables, are dietary components with an established protective role against cancer. Several molecular mechanisms are involved in the anticancer effect of polyphenols, including effects on apoptosis, cell cycle regulation, plasma membrane receptors, signaling pathways, and epigenetic mechanisms. Additionally, inhibition of glucose cellular uptake and metabolism in cancer cell lines has been described for several polyphenols, and this effect was shown to be associated with their anticarcinogenic effect. This work will review data showing an antimetabolic effect of polyphenols and its involvement in the chemopreventive/chemotherapeutic potential of these dietary compounds, in relation to breast cancer.

Assessment of the scalability of a microtiter plate system for screening of oleaginous microorganisms

Recent developments in molecular biology and metabolic engineering have resulted in a large increase in the number of strains that need to be tested, positioning high-throughput screening of microorganisms as an important step in bioprocess development. Scalability is crucial for performing reliable screening of microorganisms. Most of the scalability studies from microplate screening systems to controlled stirred-tank bioreactors have been performed so far with unicellular microorganisms. We have compared cultivation of industrially relevant oleaginous filamentous fungi and microalga in a Duetz-microtiter plate system to benchtop and pre-pilot bioreactors. Maximal glucose consumption rate, biomass concentration, lipid content of the biomass, biomass, and lipid yield values showed good scalability for Mucor circinelloides (less than 20% differences) and Mortierella alpina (less than 30% differences) filamentous fungi. Maximal glucose consumption and biomass production rates were identical for Crypthecodinium cohnii in microtiter plate and benchtop bioreactor. Most likely due to shear stress sensitivity of this microalga in stirred bioreactor, biomass concentration and lipid content of biomass were significantly higher in the microtiter plate system than in the benchtop bioreactor. Still, fermentation results obtained in the Duetz-microtiter plate system for Crypthecodinium cohnii are encouraging compared to what has been reported in literature. Good reproducibility (coefficient of variation less than 15% for biomass growth, glucose consumption, lipid content, and pH) were achieved in the Duetz-microtiter plate system for Mucor circinelloides and Crypthecodinium cohnii. Mortierella alpina cultivation reproducibility might be improved with inoculation optimization. In conclusion, we have presented suitability of the Duetz-microtiter plate system for the reproducible, scalable, and cost-efficient high-throughput screening of oleaginous microorganisms.