Lower Glucose Consumption Research Articles

Profiling biomanufactured extracellular vesicles of human forebrain spheroids in a Vertical-Wheel Bioreactor.

Extracellular vesicles (EVs) secreted by human brain cells have great potential as cell-free therapies in various diseases, including stroke. However, because of the significant amount of EVs needed in preclinical and clinical trials, EV applicationis still challenging. Vertical-Wheel Bioreactors (VWBRs) have designed features that allow for scaling up the generation of human forebrain spheroid EVs under low shear stress. In this study, EV secretion by human forebrain spheroids derived from induced pluripotent stem cells as 3D aggregates and on Synthemax II microcarriers in VWBRs were investigated with static aggregate culture as a control. The spheroids were characterized by metabolite and transcriptome analysis. The isolated EVs were characterized by nanoparticle tracking analysis, electron microscopy, and Western blot. The EV cargo was analyzed using proteomics and miRNA sequencing. The in vitro functional assays of an oxygen and glucose-deprived stroke model were conducted. Proof of concept in vivo study was performed, too. Human forebrain spheroid differentiated on microcarriers showed a higher growth rate than 3D aggregates. Microcarrier culture had lower glucose consumption per million cells and lower glycolysis gene expression but higher EV biogenesis genes. EVs from the three culture conditions showed no differences in size, but the yields from high to low were microcarrier cultures, dynamic aggregates, and static aggregates. The cargo is enriched with proteins (proteomics) and miRNAs (miRNA-seq), promoting axon guidance, reducing apoptosis, scavenging reactive oxygen species, and regulating immune responses. Human forebrain spheroid EVs demonstrated the ability to improve recovery in an in vitro stroke model and in vivo. Human forebrain spheroid differentiation in VWBR significantly increased the EV yields (up to 240-750 fold) and EV biogenesis compared to static differentiation due to the dynamic microenvironment and metabolism change. The biomanufactured EVs from VWBRs have exosomal characteristics and more therapeutic cargo and are functional in in vitro assays, which paves the way for future in vivo stroke studies.

Antitumor and immunomodulatory effects of oxygen therapy

It is well known that ischemia and hypoxia in the tumor microenvironment promote tumor progression. Оxygen deficiency in tumor microenvironment polarizes cancer cell metabolism from oxidative phosphorylation to the aerobic mode (Warburg effect) and anaerobic glycolysis. This altered carbohydrate metabolism is characterized by low energy efficiency and excessive glucose consumption. Under hypoxic conditions, the antioxidant protection of malignant cells becomes weaker, thus causing a sufficient increase of their susceptibility to direct toxic effects of reactive oxygen species (ROS). In clinical practice, oxygen saturation of tumors is usually achieved by using water-soluble ozone or hyperbaric oxygen. The ROS are shown to be produced by oxidative burst, thus being able to enhance antitumor effects of chemoradiotherapy. The immune cell-derived ROS were shown to directly inhibit tumor growth. In addition, ROS provide additional immune stimulation through the induction of mutagenesis in the tumor cells and production of immunogenic neoantigens. ROS may also enhance antitumor immune defense by inducing synthesis of interferon-γ, tumor necrosis factor-α, IL-2 and IL-6 by immune cells. Moreover, ROS may exert a negative effect on antitumor immunity. In particular, they are able to: (I) favor the recruitment and accumulation of regulatory T cells (Treg) and myeloid-derived suppressor cells (MDSCs) in the tumor microenvironment; (II) promote accumulation of alternatively activated (M2) macrophages and (N2) neutrophils, and, (III) impair presentation of immunogenic antigens (Ag) by dendritic cells. We suggest that the maximal clinical effect of oxygen therapy could be achieved in case of its simultaneous or sequential combination with immunotherapeutic interventions.The authors conclude that:– oxidative stress-induced reactive oxygen species may preferentially damage tumour cells without significantly affecting normal cells;– oxygen therapy may potentiate anti-tumour effects of chemoradiotherapy;– oxygen therapy could be effectively combined with immunotherapy to achieve maximal anticancer effects with minimal side effects.

Growth, laccase activity and role in 2,4-D degradation of Lentinus crinitus (L.) Fr. in a liquid medium

This study aimed to evaluate the role of L. crinitus laccase in the biotransformation of 2,4-D pesticide and the adsorption capacity and/or accumulation in the fungal biomass. Preliminary tests were carried out in liquid medium containing L. crinitus to detect the maximum concentration of 2,4-D in which the mycelium developed (5.025 g L−1). The growth and laccase activity for L. crinitus were analyzed using a complete factorial experimental design with variations in concentrations of glucose (16.65–33.35 g L−1), malt extract (0.825–9.175 g L−1), and 2,4-D (0.01–5 gL-1), and the mycelial samples were evaluated by SEM-EDS. The ability of L. crinitus to remove 2,4-D after 28 days was demonstrated by a decrease in concentrations of 2,4-D by up to 15.7% more than control treatments and occurred with both low (<2 g L−1) and high (>10 g L−1) glucose consumption conditions, reaching biomass concentrations below 2 g L−1, and presenting laccase activity between 3 and 6 g L−1. The laccase activity of the fungus depends on the initial amount of glucose and 2,4-D (up to 18 g L−1 and 4 g L−1). Lower concentrations of 2,4-DCP (0.01–2.70 mg L−1) may be an indicative of L. crinitus as its transformation agent. The presence of chlorine in the fungal mycelium (up to 4.0%) may represent its adsorption and/or accumulation by L. crinitus. L. crinitus can be described as a biotransformator of 2,4-D and 2,4-DCP through laccase activity, highlighting its potential for treatment of agricultural effluents.

Hexokinase 2: The preferential target of trehalose-6-phosphate over hexokinase 1.

Cancer-related metabolic features are in part maintained by hexokinase 2 upregulation, which leads to high levels of glucose-6-phosphate (G6P) and is needed to provide energy and biomass to support rapid proliferation. Using a humanized model of the yeast Saccharomyces cerevisiae, we explored how human hexokinase 2 (HK2) behaves under different nutritional conditions. At high glucose levels, yeast presents aerobic glycolysis through a regulatory mechanism known as catabolic repression, which exerts a metabolic adaptation like the Warburg effect. At high glucose concentrations, HK2 did not translocate into the nucleus and was not able to shift the metabolism toward a highly glycolytic state, in contrast to the effect of yeast hexokinase 2 (Hxk2), which is a crucial protein for the control of aerobic glycolysis in S. cerevisiae. During the stationary phase, when glucose is exhausted, Hxk2 is shuttled out of the nucleus, ceasing catabolic repression. Cells harvested at this condition display low glucose consumption rates. However, glucose-starved cells expressing HK2 had an increased capacity to consume glucose. In those cells, HK2 localized to mitochondria, becoming insensitive to G6P inhibition. We also found that the sugar trehalose-6-phosphate (T6P) is a human HK2 inhibitor, like yeast Hxk2, but was not able to inhibit human HK1, the isoform that is ubiquitously expressed in almost all mammalian tissues. In contrast to G6P, T6P inhibited HK2 even when HK2 was associated with mitochondria. The binding of HK2 to mitochondria is crucial for cancer survival and proliferation. T6P was able to reduce the cell viability of tumor cells, although its toxicity was not impressive. This was expected as cell absorption of phosphorylated sugars is low, which might be counteracted using nanotechnology. Altogether, these data suggest that T6P may offer a new paradigm for cancer treatment based on specific inhibition of HK2.

The Exploring Functional Role of Ammonium Transporters of Aspergillus oryzae in Nitrogen Metabolism: Challenges towards Cell Biomass Production.

Ammonium is a source of fermentable inorganic nitrogen essential for the growth and development of filamentous fungi. It is involved in several cellular metabolic pathways underlying nitrogen transport and assimilation. Ammonium can be transferred into the cell by an ammonium transporter. This study explored the role of ammonium transporters in nitrogen metabolism and cell biomass production in Aspergillus oryzae strain BCC 7051. Specific sequences encoding ammonium transporters (Amts) in A. oryzae were identified using genomic analysis. Four of the identified ammonium transporter genes, aoamt1-aoamt4, showed similarity in deduced amino acid sequences to the proteins in the ammonium transporter/methylammonium permease (AMT/MEP) family. Transcriptional analysis showed that the expression of aoamt2 and aoamt3 was ammonium-dependent, and was highly upregulated under ammonium-limited conditions. Their functional roles are characterized by genetic perturbations. The gene disruption and overexpression of aoamt3 indicated that the protein encoded by it was a crucial ammonium transporter associated with nitrogen metabolism and was required for filamentous growth. Compared with the wild type, the aoamt3-overexpressing strain showed superior growth performance, high biomass yield, and low glucose consumption. These results shed light on further improvements in the production of potent bioproducts by A. oryzae by manipulating the ammonium uptake capacity and nitrogen metabolism.

P-224 The sex of the human embryos affects the oxidation profile of the spent culture media

Abstract Study question Is the oxidative status of the spent culture media used in ICSI treatments related to embryo sex and newborn characteristics? Summary answer Female embryos produce higher levels of oxidative stress in the culture media than male embryos. What is known already Sex-associated differences in embryo development have previously been observed. Studies suggest that male embryos have a faster pre-implantation development, higher morphological grading and lower glucose consumption (Nasiri et al, 2018). The thermochemiluminiscence (TCL) assay quantifies the oxidative status of biological samples by catalyzing an oxidative reaction through heating and counting the photons emitted per second. TCL parameters are directly correlated to the content of oxidant agents in the sample. Previous analysis used TCL assay to relate higher oxidative metabolism in embryos with higher viability. Analysis of spent culture media with TCL could be a potential biomarker for embryo sex prediction. Study design, size, duration Prospective observational study over 86 transferred blastocysts (all single embryo transfer) that reached life-birth, belonging to ICSI treatments, including autologous and oocyte donation, performed in a single fertility clinic during three consecutive years. The oxidative status of the spent culture media was analyzed and compared with the characteristics of the newborns. Participants/materials, setting, methods Embryo cohorts were cultured in individual wells in an Embryoscope incubator, using single-step Gems culture media (Genea Biomedx). The best embryo of the cohort was selected for transfer using morphologic (ASEBIR) and morphokinetic criteria (KID Score algorithmTM, Vitrolife, (KS)). Spent culture media was analyzed in the Fertissimo TCL Analyzer (Carmel Diagnostics, Israel) and oxidation parameters were compared with the sex, weight and height of the newborns through ANOVA, C2 and Pearsons Correlation coefficient (PCC) tests. Main results and the role of chance The sex ratio of the embryos (male to female) resulted 59.3%. The sex ratio was increasingly higher the better morphological classification: C = 16.7%, B = 56.0%, A: 73.3%. KS did not give significantly different scores to the embryos depending on their sex: average KS (female)=6.78 vs average KS (male)=7.03, P = 0.621. No correlation was found between KS or the morphological classification and the weight and height of the babies: PCC (KS-height)=0.158, P = 0.349; PCC (KS-weight)=0.082, P = 0.569; average newborn height: C = 51.67±1.15cm, B = 50.57±3.48cm, A = 50.92±1.95cm, P = 0.791, average newborn weight: C = 3,386.00±459.11g, B = 3,283.35±658.12g, A = 3,229.07±732.13g, P = 0.877. TCL ratio (slope of the time curve of photon emission, representing the level of oxidant agents in the sample) was statistically significantly higher in the culture media of female embryos than of male embryos: Ratio (female)=119.54±13.37 vs Ratio (male)=111.62±15.80, P = 0.017. No statistically significant difference was found in the TCL Ratio between the three morphological classes, although the tendency of the data is towards lower levels with increasing morphological classification: Ratio (C)=120.40±14.43, ratio (B)=116.32±14.39 and ratio (A)=111.27±16.62, P = 0.237. No correlation was found between the TCL parameters and the weight and height of the newborn: PCC (Ratio-height)=-0.173, P = 0.198; PCC (Ratio-weight) 0.036, P = 0.753. Limitations, reasons for caution Patient demographics, habits and male sperm characteristics were not considered for the analysis, but might impact embryo characteristics and intrauterine development of the fetus. A larger scale study should be performed to validate results. Wider implications of the findings The differential oxidative load embryos exert over the culture medium might be related to a glucose consumption-associated sex dimorphism. Measuring the oxidative status of the culture medium could serve as a non-invasive biomarker for embryo sex selection. This technique could be an interesting option in carriers of sex-linked diseases. Trial registration number not applicable

Ac106/107 affects production of infectious progeny BV by regulating transcription of late viral genes and host cell energy metabolism.

AcMNPV is a model organism of baculovirus, and Spodoptera frugiperda is one of its hosts. Disclosing the role of ac106/107 in AcMNPV infecting Spodoptera frugiperda 9 (Sf9) cells is of great significance for modifying AcMNPV as a microbial insecticide. This work constructed recombinant baculovirus that knocking out, repairment and overexpression of ac106/107 and explored the effects of Ac106/107 on the proliferation of progeny viruses. Moreover, the potential mechanism and targets of ac106/107 were further revealed. First, compared with the Bacmid-EGFP transfection group, the progeny virus does not proliferate after knocking out of ac106/107, and the proliferation ability increases by 14.5% at 72 h post transfection (h p.t.) when overexpression of ac106/107. However, knockout, repairment and overexpression of ac106/107 have no effect on viral DNA replication. Secondly, Ac106/107-EGFP was located in the cytoplasm and nucleus. Transcription level of late viral genes and viral RNA polymerase subunit genes in the Bacmidac106/107KO -EGFP transfection group and Bacmid-Ac106/107-EGFP transfection group was reduced and increased, respectively. Thirdly, AcMNPV would increase the glucose utilization and lactate consumption of the host Sf9 cells, and Bacmidac106/107KO -EGFP transfection group had lower glucose consumption and lactic acid accumulation than Bacmid-EGFP, Bacmidac106/107KO -Ac106/107(rep)-EGFP and Bacmid-Ac106/107-EGFP transfection groups. Ac106/107 can enter the nucleus and affect transcription of viral RNA polymerase subunit genes, which in turn affects the transcription of late genes, and ultimately affects virus proliferation and energy metabolism in host cells. © 2021 Society of Chemical Industry.

The diagnostic value of lower glucose consumption for IDH1 mutated gliomas on FDG-PET

BackgroundNon-invasive diagnosis of IDH1 mutation for gliomas has great clinical significance, and PET has natural advantage to detect metabolism, as IDH mutated gliomas share lower glucose consumption.MethodsClinical data of patients with gliomas and 18F-FDG PET were retrospectively reviewed. Receiver operating characteristic curve (ROC) analysis was conducted, and standard uptake value (SUV) was estimated in combination with grades or IDH1 mutation. The glucose consumption was investigated with U251 cells expressing wild-type or mutated IDH1 by glucose assay. Quantification of glucose was determined by HPLC in clinical tissues. Meanwhile, bioinformatics and western blot were applied to analyze the expression level of metabolic enzymes (e.g. HK1, PKM2, PC) in gliomas.ResultsSeventy-one glioma cases were enrolled, including 30 carrying IDH1 mutation. The sensitivity and specificity dependent on SUVmax (3.85) predicting IDH1 mutation reached 73.2 and 86.7%, respectively. The sensitivity and specificity of differentiating grades by SUVmax (3.1) were 92.3 and 64.4%, respectively. Glucose consumption of U251 IDH1 mutant cells (0.209 ± 0.0472 mg/ml) was obviously lower than IDH1wild-type cells (0.978 ± 0.0773 mg/ml, P = 0.0001) and astrocyte controls (0.335 ± 0.0592 mg/ml, P = 0.0451). Meanwhile, the glucose quantity in IDH1mutant glioma samples were significantly lower than those in IDH1 wild-type tissues (1.033 ± 1.19608 vs 6.361 ± 4.3909 mg/g, P = 0.0051). Silico analysis and western blot confirmed that HK1 and PKM2 in IDH1 wild-type gliomas were significantly higher than in IDH1 mutant group, while PC was significantly higher in IDH1 mutant gliomas.ConclusionSUVmax on PET can predict IDH1 mutation with adequate sensitivity and specificity, as is supported by reduced glucose consumption in IDH1 mutant gliomas.

Toxicity of topically applied drugs beyond skin irritation: Static skin model vs. Two organs-on-a-chip.

Skin model cultivation under static conditions limits the observation of the toxicity to this single organ. Biology-inspired microphysiological systems associating skin with a liver in the same circulating medium provide a more comprehensive insight into systemic substance toxicity; however, its advantages or limitations for topical substance toxicity remain unknown. Herein, we performed topical (OECD test guideline no. 439) and systemic administration of terbinafine in reconstructed human skin (RHS) vs. a RHS plus liver model cultured in TissUse' HUMIMIC Chip2 (Chip2). Aiming for a more detailed insight into the cutaneous substance irritancy/toxicity, we assessed more than the MTT cell viability: lactate dehydrogenase (LDH), lactate and glucose levels, as well as inherent gene expressions. Sodium dodecyl sulfate (SDS) was the topical irritant positive control. We confirmed SDS irritancy in both static RHS and Chip2 culture by the damage in the morphology, reduction in the lactate production and lower glucose consumption. In the static RHS, the SDS-treated tissues also released significantly high LDH (82%; p<0.05) and significantly lower IL-6 release (p<0.05), corroborating with the other metabolic levels. In both static RHS and Chip2 conditions, we confirmed absence of irritancy or systemic toxicity by LDH, glucose or lactate levels for topical 1% and 5% terbinafine and systemic 0.1% terbinafine treatment. However, topical 5% terbinafine treatment in the Chip2 upregulated IL-1α in the RHS, unbalanced apoptotic and proliferative cell ratios in the liver and significantly increased its expression of CYP1A2 and 3A4 enzymes (p<0.05), proving that it has passed the RHS barrier promoting a liver impact. Systemic 0.1% terbinafine treatment in the Chip2 increased RHS expression of EGFR, increased apoptotic cells in the liver, downregulated liver albumin expression and upregulated CYP2C9 significantly (p<0.05), acting as an effective hepatotoxic terbinafine control. The combination of the RHS and liver model in the Chip2 allowed a more sensitive assessment of skin and hepatic effects caused by chemicals able to pass the skin (5% terbinafine and SDS) and after systemic 0.1% terbinafine application. The present study opens up a more complex approach based on the microphysiological system to assess more than a skin irritation process.

Sex hormone-binding globulin regulates glucose metabolism in human placental trophoblasts via cAMP/PKA/CREB1.

AimThis study was conducted to investigate the effects of sex hormone‐binding globulin (SHBG) on glucose metabolism and insulin resistance in human placental trophoblasts and involvement of the cAMP/PKA/CREB1 signaling pathway in these effects.MethodsAn insulin resistance cell model of human trophoblasts was established. An SHBG‐overexpression plasmid was transfected into these cells, and the expression of glucose transporter 1 (GLUT1), CREB and p‐CREB was detected and analyzed in normal cells, model cells and all groups of transfected cells by real‐time PCR and western blotting; cAMP, PKA, glucose consumption and pyruvic acid levels were also detected.ResultsAmong the four groups, there was no significant difference in the expression of CREB mRNA or GLUT1 mRNA (P > 0.05); however, CREB, p‐CREB, GLUT1 protein, cAMP and PKA showed low expression (P < 0.05) and cell glucose consumption and pyruvate production were decreased (P < 0.05) in the model group, compared to the normal group. SHBG overexpression in insulin‐resistant cells partially increased the levels of p‐CREB, GLUT1, cAMP and PKA (P < 0.05). Intracellular glucose consumption and pyruvate production were nearly restored to the levels observed in cells from the normal group.ConclusionSex hormone‐binding globulin regulates GLUT1 expression via the cAMP/PKA/CREB1 pathway and affects glucose transport in the placenta, which can induce insulin resistance and gestational diabetes.

Salmonella enterica Optimizes Metabolism After Addition of Acyl-Homoserine Lactone Under Anaerobic Conditions

Acyl-homoserine lactones (AHLs) are quorum sensing (QS) signaling molecules that mediate cell-to-cell communication in Gram-negative bacteria. Salmonella does not produce AHL, however, it can recognize AHLs produced by other species through SdiA protein modulating important cellular functions. In this work, the influence of the N-dodecanoyl-DL-homoserine lactone (C12-HSL) on glucose consumption, metabolic profile, and gene expression of Salmonella throughout the cultivation time in Tryptic Soy Broth (TSB) under anaerobic conditions was evaluated. Analysis of the supernatant culture in high-performance liquid chromatography (HPLC) revealed lower glucose uptake after 4 and 6 h of the addition of C12-HSL. Gas chromatography-mass spectrometry (GC-MS) based analysis of the intracellular metabolites revealed C12-HSL perturbation in the abundance levels of metabolites related to the metabolic pathways of glycerolipids, purines, amino acids, and aminoacyl-tRNA biosynthesis. The real-time quantitative PCR (RT-qPCR) indicated that Salmonella increase expression of genes associated with nucleoside degradation and quantification of metabolites supported the induction of pentose phosphate pathway to ensure growth under lower glucose consumption. The obtained data suggest an important role of C12-HSL in the optimization of metabolism at a situation of high population densities.

Administration of an Immune Stimulant during the Transition Period Improved Lipid Metabolism and Rumination without Affecting Inflammatory Status.

Simple SummaryImmune stimulants are widely used to address immune dysfunctions that occur in transitioning dairy cows, reducing the likelihood they will develop infectious diseases. This study elucidates the effectiveness of an immune stimulant in promoting rumination recovery, reducing lipid mobilization and ketogenesis, and affecting the levels of circulating antioxidant systems in early lactation. These findings highlight the stimulant’s potential effect in treating metabolic disorders of the transition period in dairy cows.Omnigen-AF (OAF) increases leukocyte functions in immunosuppressed animal models and reduces incidence of infectious diseases in early lactating dairy cows, although its mode of action is still unclear. This study aims to provide a wider perspective of the metabolic effect of OAF to test its potential as a strategy to address metabolic disorders of the transition period. A group of 10 Holstein dairy cows were divided into 2 groups: The treated group (IMS; 5 cows) received 32.5 g of OAF twice a day (65 g d−1) as top-dress in the morning and afternoon feeds from −55 to 42 days from calving (DFC), whereas the control group (CTR; 5 cows) received no supplementation. From −62 to 42 DFC, body condition score, body weight, dry matter intake, rumination time and milk yield were measured; blood samples were collected weekly to assess a wide hematochemical profile and to test white blood cell functions by ex-vivo challenge assays. At 30 DFC, rumen fluid was collected and analyzed for pH, volatile fatty acids composition, urea nitrogen, and lactate contents. Data were submitted to ANOVA using a mixed model for repeated measures, including treatment, time, and their interaction as fixed effects. OAF decreased blood nonesterified fatty acids and beta hydroxybutyrate concentrations and increased rumination time in early lactation. Leukocytes from IMS cows had lower lactate production and lower glucose consumption after ex-vivo stimulation. OAF did not reduce the acute phase response indicators and reduced the blood concentrations of albumin and antioxidants after calving, suggesting impairment of hepatic functions related to protein synthesis and antioxidant management. Nevertheless, the lack of effect on bilirubin and liver enzymes refutes the possibility of severe liver damage occurring with OAF supplementation. Positive effects in reducing mobilization of body fats and ketogenesis and in increasing rumination time after calving suggest OAF effectiveness in preventing metabolic disorders of the transition period.

Adaptive Responses in the Metabolism of &lt;i&gt;Escherichia coli&lt;/i&gt; in View of Gene Expressions under Aerobic and Micro-aerobic Condition

The major aims of biology to understanding life at a systems level. Escherichia coli is a metabolically versatile bacterium able to respond to changes in environmental factors availability. The effect of pH downshift on fermentation characteristics was investigated in a continuous culture of Escherichia coli at aerobic and micro-aerobic conditions. Regardless of oxygen availability, higher levels of acetate were associated with lower biomass yields and lower glucose consumption rates at pH 5.5 as compared to the observations made at pH 7.0. Observed gene expressions indicated that the down- regulation of the glucose uptake rate corresponded to the down-regulation of ptsG gene expression which in turn was caused by the up-regulation of mlc gene under the positive control of Crp. In accordance with up-regulation of arcA gene expression at acidic conditions, the expressions of TCA cycle-related genes such as icdA and gltA, and the respiratory chain gene cyoA were down-regulated, whereas cydB gene expression was up-regulated. Decreased activity of the TCA cycle caused more acetate formation at lower pH levels. Under micro-aerobic condition, higher levels of formate and lactate were produced at lower pH due to up-regulation of pflA, yfiD and ldhA genes. Meanwhile, lower levels of ethanol were produced due to the down-regulation of adhE gene at lower pH, as compared to the observation at neutral pH. The combined effect of pH and temperature on gene expression was also investigated and observed that decreases in the specific glucose consumption rate were associated with increases in the specific acetate production rate. This type of information is useful for the production of recombinant proteins, bio-molecules, simultaneous saccharification and fermentation (SSF) and strain improvement.

Overexpression of Pyruvate Dehydrogenase E1α Subunit Inhibits Warburg Effect and Induces Cell Apoptosis Through Mitochondria-Mediated Pathway in Hepatocellular Carcinoma.

Most cancers rely disproportionately on glycolysis for energy even in the presence of an adequate oxygen supply, a condition known as “aerobic glycolysis,” or the “Warburg effect.” Pyruvate dehydrogenase E1α subunit (PDHA1) is one of the main factors for the metabolic switch from oxidative phosphorylation (OXPHOS) to aerobic glycolysis and has been suggested to be closely associated with tumorigenesis. Here we observed that the PDHA1 protein was reduced in hepatocellular carcinoma (HCC) specimens by immunohistochemistry and Western blot, which was significantly associated with poor overall survival. To further analyze the function of PDHA1 in cancer cells, PDHA1 was upregulated in the HCC cell lines SMMC-7721 and HepG2. The results demonstrated that overexpression of the PDHA1 gene inhibited aerobic glycolysis with lower lactate via increased PDH activity; meanwhile, mitochondrial OXPHOS was enhanced accompanied with higher ATP and lower glucose consumption. We also found that apoptosis was promoted and intrinsic pathway proteins were increased in PDHA1-overexpressing cells. Collectively, our data indicate that reduced PDHA1 protein expression is associated with the poor clinical outcome of HCC. Upregulated PDHA1 gene expression can inhibit the Warburg effect and enhance the mitochondria-mediated apoptosis pathway.

Obligatory role of endoplasmic reticulum in brain FDG uptake.

The endoplasmic reticulum (ER) contains hexose-6P-dehydrogenase (H6PD). This enzyme competes with glucose-6P-phosphatase for processing a variety of phosphorylated hexoses including 2DG-6P. The present study aimed to verify whether this ER glucose-processing machinery contributes to brain FDG uptake. Effect of the H6PD inhibitor metformin on brain 18F-FDG accumulation was studied, in vivo, by microPET imaging. These data were complemented with the in vitro estimation of the lumped constant (LC). Finally, reticular accumulation of the fluorescent 2DG analogue 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxyglucose (2NBDG) and its response to metformin was studied by confocal microscopy in cultured neurons and astrocytes. Metformin halved brain 18F-FDG accumulation without altering whole body tracer clearance. Ex vivo, this same response faced the doubling of both glucose consumption and lactate release. The consequent fall in LC was not explained by any change in expression or activity of its theoretical determinants (GLUTs, hexokinases, glucose-6P-phosphatase), while it agreed with the drug-induced inhibition of H6PD function. In vitro, 2NBDG accumulation selectively involved the ER lumen and correlated with H6PD activity being higher in neurons than in astrocytes, despite a lower glucose consumption. The activity of the reticular enzyme H6PD profoundly contributes to brain 18F-FDG uptake. These data challenge the current dogma linking 2DG/FDG uptake to the glycolytic rate and introduce a new model to explain the link between 18-FDG uptake and neuronal activity.

Influence of rice straw-derived dissolved organic matter on lactic acid fermentation by Rhizopus oryzae

Rice straw can be used as carbon sources for lactic acid fermentation. However, only a small amount of lactic acid is produced even though Rhizopus oryzae can consume glucose in rice straw-derived hydrolysates. This study correlated the inhibitory effect of rice straw with rice straw-derived dissolved organic matter (DOM). Lactic acid fermentations with and without DOM were conducted to investigate the effect of DOM on lactic acid fermentation by R.oryzae. Fermentation using control medium with DOM showed a similar trend to fermentation with rice straw-derived hydrolysates, showing that DOM contained the major inhibitor of rice straw. DOM assay indicated that it mainly consisted of polyphenols and polysaccharides. The addition of polyphenols and polysaccharides derived from rice straw confirmed that lactic acid fermentation was promoted by polysaccharides and significantly inhibited by polyphenols. The removal of polyphenols also improved lactic acid production. However, the loss of polysaccharides during the removal of polyphenols resulted in low glucose consumption. This study is the first to investigate the effects of rice straw-derived DOM on lactic acid fermentation by R.oryzae. The results may provide a theoretical basis for identifying inhibitors and promoters associated with lactic acid fermentation and for establishing suitable pretreatment methods.

Improvement of pyruvate production based on regulation of intracellular redox state in engineered Escherichia coli

The commercial demand for pyruvate has been expanding. However, some challenges need to be overcome in the microbial production of pyruvate, such as low glucose consumption caused by excessive accumulation of NADH. In this study, weakening or block of the TCA cycle, overexpression of foreign NADH oxidase, and carbon sources with different oxidation state was attempted to decrease NADH accumulation in engineered strain YP211. Results showed that blocking or weakening TCA cycle could not lower the intracellular redox state in strain YP211.Overexpressing NADH oxidase from Lactococcus lactis significantly decreased the intracellular NADH content and increased the consumption rate of glucose. However, the yield of pyruvate did not increase significantly. Compared with glucose as carbon source, sodium gluconate with a higher oxidation state resulted in a significant decrease of NADH/NAD+, and the concentration and yield of pyruvate increased by 62 and 6%, respectively. In the fed-batch fermentation, the yield of pyruvate increased to 0.78 g/g gluconate, and the concentration of pyruvate reached 78.8 g/L. It was suggested that sodium gluconate was a more ideal carbon source for strain YP211, which could effectively decrease NADH content and improve the pyruvate production.

Escherichia coli expressing endoglucanase gene from Thai higher termite bacteria for enzymatic and microbial hydrolysis of cellulosic materials

Background: Endoglucanase plays a major role in initiating cellulose hydrolysis. Various wild-type strains were searched to produce this enzyme but mostly low extracellular enzyme activities were obtained. To improve extracellular enzyme production for potential industrial applications, endoglucanase gene of Bacillus subtilis M015, isolated from Thai higher termite, was expressed in a periplasmic-leaky Escherichia coli . Then, the use of crude recombinant endoglucanase (EglS) with a commercial cellulase (Cel) for hydrolyzing celluloses and microbial hydrolysis using whole bacterial cells was evaluated. Results: E. coli Glu5 expressing endoglucanase at high levels was successfully constructed. It produced EglS (55 kDa) with extracellular activity of 18.56 U/mg total protein at optimal hydrolytic conditions (pH 4.8 and 50oC). EglS was highly stable (over 80% activity retained) at 40-50oC after 100 h. The addition of EglS significantly improved the initial sugar production rates of Cel on the hydrolysis of carboxymethyl cellulose (CMC), microcrystalline cellulose and corncob about 5.2, 1.7, and 4.0 folds, respectively, as compared to those with Cel alone. E. coli Glu5 could secrete EglS with high activity in the presence of glucose (1% w/v) and Tween 80 (5% w/v) with low glucose consumption. Microbial hydrolysis of CMC using E. coli Glu5 yielded 26 mg reducing sugar/g CMC at pH 7.0 and 37oC after 48 h. Conclusions: The recombinant endoglucanase activity was improved by 17 times compared with that of the native strain, and could greatly enhance the enzymatic hydrolysis of all studied celluloses when combined with a commercial cellulase. Normal 0 false false false EN-US X-NONE X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:Tabla normal; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin:0cm; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:10.0pt; font-family:Times New Roman,serif;}

Lectin PCL inhibits the Warburg effect of PC3 cells by combining with EGFR and inhibiting HK2.

Prostatic carcinoma is the most aggressive tumor in adult men. Warburg effect is an important characteristic of tumor cell metabolism including prostate cancer cells, in which hexokinase 2 (HK2), a major rate-limiting enzyme involved in Warburg effect, is selectively upregulated. The lectin PCL is a mannose binding lectin which induces tumor cell apoptosis and autophagy. In the present study, we report that PCL could lower glucose consumption and lactate production, shift the Warburg effect by inhibiting the expression of HK2 in PC3 cells and the suppression of HK2 by siRNA reversed the effect of PCL on glucose consumption and lactate production. The expressionof HK2 is closely related to epidermal growth factor receptor (EGFR) and downstream signaling pathway activation, therefore, we investigated the interaction of PCL with EGFR by western blot analysis and found that PCL could suppress the binding of epidermal growth factor (EGF) with EGFR and HK2 expression. Also, we explored the binding mechanism between the PCL and EGFR through molecular docking and molecular dynamics simulations and found that PCL bocked the active site of EGFR which is also the binding site of the nature ligand EGF, the resulting conformation has higher stability than EGF in complex with EGFR. The results indicated that PCL could competitively bind to EGFR binding pocket and then prevent EGF from binding to EGFR, blocking the autophosphorylation of the EGFR tyrosine kinase, after that the EGFR activation is inhibited. Collectively, our studies concluded that PCL inhibits tumor cell glycolysis by combining with EGFR and reducing HK2 expression.

Increased glucose utilization and cell growth of Corynebacterium glutamicum by modifying the glucose-specific phosphotransferase system (PTSGlc) genes.

The phosphoenolpyruvate:glucose phosphotransferase system (PTSGlc) is the major pathway of glucose uptake in Corynebacterium glutamicum. This study investigated glucose consumption rate, cell growth, and metabolite changes resulting from modification of PTSGlc. The classical l-lysine producer C. glutamicum XQ-8 exhibited low glucose consumption, cell growth, and l-lysine production rates, whereas these parameters were significantly increased during cultivating on glucose plus maltose, through inactivation of SugR, or by overexpression of PTSGlc genes. XQ-8sugR::cat/pDXW-8-ptsI exhibited the highest increase in glucose consumption, growth rate, and l-lysine production, followed by XQ-8sugR::cat/pDXW-8-ptsG. However, overexpression of ptsH had little effect on the above-mentioned factors. Although co-overexpression of ptsGHI led to the highest glucose consumption, growth rate, and final l-lysine production; the l-lysine production rate was lower than that of XQ-8sugR::cat/pDXW-8-ptsIH. In fed-batch fermentation, XQ-8sugR::cat/pDXW-8-ptsIH had a higher growth rate of 0.54 h-1 to a dry cell mass of 66 g·L-1 after 16 h, and had a higher l-lysine production rate of 159.2 g·L-1 after 36 h. These results indicate that modification of the sugar transport systems improves amino acid production, especially for mutants obtained by repeated physical and (or) chemical mutagenesis. However, modification of these systems needs to be performed on a case-by-case basis.