TP53-Induced Glycolysis and Apoptosis Regulator (TIGAR) Is Upregulated in Lymphocytes Stimulated with Concanavalin A

The glycolytic modulator TP53-Inducible Glycolysis and Apoptosis Regulator (TIGAR) is overexpressed in several types of cancer and has a role in metabolic rewiring during tumor development. However, little is known about the role of this enzyme in proliferative tissues under physiological conditions. In the current work, we analysed the role of TIGAR in primary human lymphocytes stimulated with the mitotic agent Concanavalin A (ConA). We found that TIGAR expression was induced in stimulated lymphocytes through the PI3K/AKT pathway, since Akti-1/2 and LY294002 inhibitors prevented the upregulation of TIGAR in response to ConA. In addition, suppression of TIGAR expression by siRNA decreased the levels of the proliferative marker PCNA and increased cellular ROS levels. In this model, TIGAR was found to support the activity of glucose 6-phosphate dehydrogenase (G6PDH), the first enzyme of the pentose phosphate pathway (PPP), since the inhibition of TIGAR reduced G6PDH activity and increased autophagy. In conclusion, we demonstrate here that TIGAR is upregulated in stimulated human lymphocytes through the PI3K/AKT signaling pathway, which contributes to the redirection of the carbon flux to the PPP.

Induced expression of the zwf gene in the presence of glucose contributes to lowering of glucose 6-phosphate level and consequently reduction of growth rate of Mycobacterium smegmatis

In Mycobacterium smegmatis (renamed Mycolicibacterium smegmatis ), glucose 6-phosphate (G6P) level is exceptionally high as compared to other bacteria, E. coli for example. Earlier investigations have indicated that G6P protects M. smegmatis (Msm) against oxidative stress-inducing agents. G6P is a glycolytic intermediate formed either directly through the phosphorylation of glucose or indirectly via the gluconeogenic pathway. Its consumption is catalysed by several enzymes, one of which being the NADPH dependent G6P dehydrogenase (G6PDH) encoded by zwf (msmeg_0314). While investigating the extent to which the carbon sources glucose and glycerol influence Msm growth, we observed that intracellular concentration of G6P was lower in the former’s presence than the latter. We could correlate this difference with that in the growth rate, which was higher in glycerol than glucose. We also found that lowering of G6P content in glucose-grown cells was triggered by the induced expression of zwf and the resultant increase in G6PDH activity. When we silenced zwf using CRISPR-Cas9 technology, we observed a significant rise in the growth rate of Msm. Therefore, we have found that depletion of G6P in glucose-grown cells due to increased G6PDH activity is at least one reason why the growth rate of Msm in glucose is less than glycerol. However, we could not establish a similar link-up between slow growth in glucose and lowering of G6P level in the case of Mycobacterium tuberculosis (Mtb). Mycobacteria, therefore, may have evolved diverse mechanisms to ensure that they use glycerol preferentially over glucose for their growth.

Transcriptional profile of bovine preimplantation development selected based on G6PDH activity

The oocyte is the female gamete that contributes not only half of the genetic material but also all of the cytoplasm to the zygote, supplying the transcripts, proteins, mitochondria and other components necessary for early embryonic development. The intrinsic oocyte quality is one of the main factors affecting the embryo yield, the implantation rate and the rate of healthy offspring. It is obvious that a fertilized oocyte must reach the blastocyst stage within 6–9 days in the proper culture conditions to have a significant chance of inducing a pregnancy and producing an offspring. The ability to sustain the first week of embryonic development is clearly influenced by the follicular status from which the oocyte is obtained indicating that this developmental potential is inherent within certain oocytes. Since most early embryos that do not reach the blastocyst stage are blocked at or close to the maternal to zygotic transition (MZT)-stage, which occurs at the eight-cell stage in cattle, one could speculate that incompetent oocytes fail to appropriately activate the embryonic genome. Oocyte selection based on glucose-6-phosphate dehydrogenase (G6PDH) activity has been successfully used to differentiate between competent and incompetent bovine oocytes. Recently, molecular regulation of genes regulating biological process of Brilliant Cresyl Blue staining (BCB) selected oocytes and embryos was investigated to explain their variation in quality and developmental potentiality. This short review will highlights some of these efforts that have been done in this interesting area of research.

BIMG-02. IMAGING IMMORTALITY: TERT EXPRESSION ALTERS GLUCOSE METABOLISM IN LOW-GRADE GLIOMAS IN A MANNER THAT CAN BE LEVERAGED FOR NONINVASIVE METABOLIC IMAGING

Telomerase reverse transcriptase (TERT) is essential for tumor immortality and uncontrolled proliferation, including in low-grade oligodendrogliomas (LGOGs). Since it is silenced in somatic cells, TERT is also a therapeutic target. Non-invasive imaging of TERT can differentiate tumor from normal brain or lesions such as gliosis and allow assessment of response to therapy. The goal of this study was to identify magnetic resonance spectroscopy (MRS)-detectable metabolic alterations associated with TERT that can be leveraged for noninvasive imaging in LGOGs. We examined patient-derived BT54 neurospheres in which TERT expression was silenced by RNA interference. 1H-MRS showed that steady-state levels of NAD(P)/H, glutathione, aspartate and AXP were elevated in BT54TERT+ neurospheres relative to BT54TERT-. Glucose flux through the pentose phosphate pathway (PPP) is essential for generating NADPH, which maintains glutathione homeostasis. 13C-MRS confirmed that [2-13C]-glucose flux through the PPP was elevated in BT54TERT+ neurospheres relative to BT54TERT-, an effect associated with higher activity of the PPP enzyme glucose-6-phosphate dehydrogenase (G6PDH). Hyperpolarized 13C-MRS is a method of increasing the signal to noise ratio of 13C-MRS such that it can monitor metabolic fluxes noninvasively in cells, animals and patients. Consistent with elevated PPP flux and G6PDH activity, hyperpolarized [U-13C]-glucose metabolism via the PPP to 6-phosphogluconate (6-PG) was elevated in BT54TERT+ neurospheres relative to BT54TERT-. Importantly, examination of an additional patient-derived LGOG model, the SF10417 model which readily forms orthotopic tumor xenografts in rats, showed that 6-PG production from hyperpolarized [U-13C]-glucose demarcated tumor from normal brain. Furthermore, LGOG patient biopsies had elevated NAD(P)/H, glutathione, aspartate, AXP and G6PDH activity relative to gliosis biopsies, confirming the clinical validity of our observations. Collectively, we have identified a metabolic signature of TERT expression that can be leveraged via hyperpolarized [U-13C]-glucose to improve diagnosis and treatment response monitoring for LGOG patients.

Cytoplasmic glucose-6-phosphate dehydrogenase plays an important role in the silicon-enhanced alkaline tolerance in highland barley

Glucose-6-phosphate dehydrogenase (G6PDH), as a key enzyme in the pentose phosphate pathway, extensively responds to the biotic and abiotic stresses. In this study we focussed on the G6PDH role in the alleviation of alkaline stress induced by silicon (Si) in highland barley. Application of Si reduced the water loss and malondialdehyde (MDA) and reactive oxygen species (ROS) contents, improved the fresh weight, photosynthesis, K+ content, and the superoxide dismutase (SOD) and catalase (CAT) activities, thus alleviating the damage caused by alkaline stress. The G6PDH activity, especially the cytoplasmic G6PDH, significantly increased under alkaline stress, and was further stimulated by the addition of exogenous Si. Meanwhile, the levels of NADPH and reduced glutathione (GSH) showed similar profiles to G6PDH activity under NaHCO3 and NaHCO3 + Si treatments. The inhibition of G6PDH activity by glucosamine abolished the relieving effect of Si on alkaline stress, which was manifested in the increase of ROS and the decrease of GSH content. Together, our results suggest that Si-enhanced tolerance of alkaline stress may be related to the regulation of GSH levels by the cytoplasmic G6PDH in highland barley.

The specific PKC-α inhibitor chelerythrine blunts costunolide-induced eryptosis

Costunolide, a natural sesquiterpene lactone, has multiple pharmacological activities such as neuroprotection or induction of apoptosis and eryptosis. However, the effects of costunolide on pro-survival factors and enzymes in human erythrocytes, e.g. glutathione and glucose-6-phosphate dehydrogenase (G6PDH) respectively, have not been studied yet. Our aim was to determine the mechanisms underlying costunolide-induced eryptosis and to reverse this process. Phosphatidylserine exposure was estimated from annexin-V-binding, cell volume from forward scatter in flow cytometry, and intracellular glutathione [GSH]i from high performance liquid chromatography. The oxidized status of intracellular glutathione and enzyme activities were measured by spectrophotometry. Treatment of erythrocytes with costunolide dose-dependently enhanced the percentage of annexin-V-binding cells, decreased the cell volume, depleted [GSH]i and completely inhibited G6PDH activity. The effects of costunolide on annexin-V-binding and cell volume were significantly reversed by pre-treatment of erythrocytes with the specific PKC-α inhibitor chelerythrine. The latter, however, had no effect on costunolide-induced GSH depletion. Costunolide induces eryptosis, depletes [GSH]i and inactivates G6PDH activity. Furthermore, our study reveals an inhibitory effect of chelerythrine on costunolide-induced eryptosis, indicating a relationship between costunolide and PKC-α. In addition, chelerythrine acts independently of the GSH depletion. Understanding the mechanisms of G6PDH inhibition accompanied by GSH depletion should be useful for development of anti-malarial therapeutic strategies or for synthetic lethality-based approaches to escalate oxidative stress in cancer cells for their sensitization to chemotherapy and radiotherapy.

Elevated CO2 improves both lipid accumulation and growth rate in the glucose-6-phosphate dehydrogenase engineered Phaeodactylum tricornutum

Background Numerous studies have shown that stress induction and genetic engineering can effectively increase lipid accumulation, but lead to a decrease of growth in the majority of microalgae. We previously found that elevated CO2 concentration increased lipid productivity as well as growth in Phaeodactylum tricornutum, along with an enhancement of the oxidative pentose phosphate pathway (OPPP) activity. The purpose of this work directed toward the verification of the critical role of glucose-6-phosphate dehydrogenase (G6PDH), the rate-limiting enzyme in the OPPP, in lipid accumulation in P. tricornutum and its simultaneous rapid growth rate under high-CO2 (0.15%) cultivation. Results In this study, G6PDH was identified as a target for algal strain improvement, wherein G6PDH gene was successfully overexpressed and antisense knockdown in P. tricornutum, and systematic comparisons of the photosynthesis performance, algal growth, lipid content, fatty acid profiles, NADPH production, G6PDH activity and transcriptional abundance were performed. The results showed that, due to the enhanced G6PDH activity, transcriptional abundance and NAPDH production, overexpression of G6PDH accompanied by high-CO2 cultivation resulted in a much higher of both lipid content and growth in P. tricornutum, while knockdown of G6PDH greatly decreased algal growth as well as lipid accumulation. In addition, the total proportions of saturated and unsaturated fatty acid, especially the polyunsaturated fatty acid eicosapentaenoic acid (EPA; C20:5, n-3), were highly increased in high-CO2 cultivated G6PDH overexpressed strains. Conclusions The successful of overexpression and antisense knockdown of G6PDH well demonstrated the positive influence of G6PDH on algal growth and lipid accumulation in P. tricornutum. The improvement of algal growth, lipid content as well as polyunsaturated fatty acids in high-CO2 cultivated G6PDH overexpressed P. tricornutum suggested this G6PDH overexpression-high CO2 cultivation pattern provides an efficient and economical route for algal strain improvement to develop algal-based biodiesel production.

The relationship between carbon and nitrogen metabolism in cucumber leaves acclimated to salt stress

The study examines the effect of acclimation on carbon and nitrogen metabolism in cucumber leaves subjected to moderate and severe NaCl stress. The levels of glucose, sucrose, NADH/NAD+-GDH, AspAT, AlaAT, NADP+-ICDH, G6PDH and 6GPDH activity were determined after 24 and 72 hour periods of salt stress in acclimated and non-acclimated plants. Although both groups of plants showed high Glc and Suc accumulation, they differed with regard to the range and time of accumulation. Acclimation to salinity decreased the activities of NADP+-ICDH and deaminating NAD+-GDH compared to controls; however, these enzymes, together with the other examined parameters, showed elevated values in the stressed plants. The acclimated plants showed higher G6PDH activity than the non-acclimated plants, whereas both groups demonstrated similar 6PGDH activity. The high activities of NADH-GDH, AlaAT and AspAT observed in the examined plants could be attributed to a high demand for glutamate. The observed changes may be required for the maintenance of correct TCA cycle activity, and acclimation appeared to positively influence these adaptive processes.

Thioredoxin regulates G6PDH activity by changing redox states of OpcA in the nitrogen-fixing cyanobacterium Anabaena sp. PCC 7120

Glucose 6-phosphate dehydrogenase (G6PDH) catalyzes the first reaction in the oxidative pentose phosphate pathway. In green plant chloroplasts, G6PDH is a unique redox-regulated enzyme, since it is inactivated under the reducing conditions. This regulation is accomplished using a redox-active cysteine pair, which is conserved in plant G6PDH. The inactivation of this enzyme under conditions of light must be beneficial to prevent release of CO2 from the photosynthetic carbon fixation cycle. In the filamentous, heterocyst-forming, nitrogen-fixing cyanobacterium Anabaena sp. PCC 7120 (Anabaena 7120), G6PDH plays a pivotal role in providing reducing power for nitrogenase, and its activity is also reported to be suppressed by reduction, though Anabaena G6PDH does not conserve the critical cysteines for regulation. Based on the thorough analyses of the redox regulation mechanisms of G6PDH from Anabaena 7120 and its activator protein OpcA, we found that m-type thioredoxin regulates G6PDH activity by changing the redox states of OpcA. Mass spectrometric analysis and mutagenesis studies indicate that Cys393 and Cys399 of OpcA are responsible for the redox regulation property of this protein. Moreover, in vivo analyses of the redox states of OpcA showed that more than half of the OpcA is present as an oxidized form, even under conditions of light, when cells are cultured under the nitrogen-fixing conditions. This redox regulation of OpcA might be necessary to provide reducing power for nitrogenase by G6PDH in heterocysts even during the day.