Concentration Of Glucose 6-phosphate Research Articles

1H and 31P magnetic resonance spectroscopy reveals potential pathogenic and biomarker metabolite alterations in Lafora disease.

Lafora disease is a fatal teenage-onset progressive myoclonus epilepsy and neurodegenerative disease associated with polyglucosan bodies. Polyglucosans are long-branched and as a result precipitation- and aggregation-prone glycogen. In mouse models, downregulation of glycogen synthase, the enzyme that elongates glycogen branches, prevents polyglucosan formation and rescues Lafora disease. Mouse work, however, has not yet revealed the mechanisms of polyglucosan generation, and few in vivo human studies have been performed. Here, non-invasive in vivo magnetic resonance spectroscopy (1H and 31P) was applied to test scan feasibility and assess neurotransmitter balance and energy metabolism in Lafora disease towards a better understanding of pathogenesis. Macromolecule-suppressed gamma-aminobutyric acid (GABA)-edited 1H magnetic resonance spectroscopy and 31P magnetic resonance spectroscopy at 3 and 7 tesla, respectively, were performed in 4 Lafora disease patients and a total of 21 healthy controls (12 for the 1H magnetic resonance spectroscopy and 9 for the 31PMRS). Spectra were processed using in-house software and fit to extract metabolite concentrations. From the 1H spectra, we found 33% lower GABA concentrations (P = 0.013), 34% higher glutamate + glutamine concentrations (P = 0.011) and 24% lower N-acetylaspartate concentrations (P = 0.0043) in Lafora disease patients compared with controls. From the 31P spectra, we found 34% higher phosphoethanolamine concentrations (P = 0.016), 23% lower nicotinamide adenine dinucleotide concentrations (P = 0.003), 50% higher uridine diphosphate glucose concentrations (P = 0.004) and 225% higher glucose 6-phosphate concentrations in Lafora disease patients versus controls (P = 0.004). Uridine diphosphate glucose is the substrate of glycogen synthase, and glucose 6-phosphate is its extremely potent allosteric activator. The observed elevated uridine diphosphate glucose and glucose 6-phosphate levels are expected to hyperactivate glycogen synthase and may underlie the generation of polyglucosans in Lafora disease. The increased glutamate + glutamine and reduced GABA indicate altered neurotransmission and energy metabolism, which may contribute to the disease's intractable epilepsy. These results suggest a possible basis of polyglucosan formation and potential contributions to the epilepsy of Lafora disease. If confirmed in larger human and animal model studies, measurements of the dysregulated metabolites by magnetic resonance spectroscopy could be developed into non-invasive biomarkers for clinical trials.

Deglycation activity of the Escherichia coli glycolytic enzyme phosphoglucose isomerase

Glycation is a spontaneous chemical reaction, which affects the structure and function of proteins under normal physiological conditions. Therefore, organisms have evolved diverse mechanisms to combat glycation. In this study, we show that the Escherichia coli glycolytic enzyme phosphoglucose isomerase (Pgi) exhibits deglycation activity. We found that E. coli Pgi catalyzes the breakdown of glucose 6-phosphate (G6P)-derived Amadori products (APs) in chicken lysozyme. The affinity of Pgi to the glycated lysozyme (Km, 1.1 mM) was ten times lower than the affinity to its native substrate, fructose 6-phosphate (Km, 0.1 mM). However, the high kinetic constants of the enzyme with the glycated lysozyme (kcat, 396 s−1 and kcat/Km, 3.6 × 105 M−1 s−1) indicated that the Pgi amadoriase activity may have physiological implications. Indeed, when using total E. coli protein (20 mg/mL) as a substrate in the deglycation reaction, we observed a release of G6P from the bacterial protein at a Pgi specific activity of 33 μmol/min/mg. Further, we detected 11.4 % lower APs concentration in protein extracts from Pgi-proficient vs. deficient cells (p = 0.0006) under conditions where the G6P concentration in Pgi-proficient cells was four times higher than in Pgi-deficient cells (p = 0.0001). Altogether, these data point to physiological relevance of the Pgi deglycation activity.

Metabolomics profiling reveals differences in proliferation between tumorigenic and non-tumorigenic Madin-Darby canine kidney (MDCK) cells.

Madin-Darby canine kidney (MDCK) cells are a cellular matrix in the production of influenza vaccines. The proliferation rate of MDCK cells is one of the critical factors that determine the vaccine production cycle. It is yet to be determined if there is a correlation between cell proliferation and alterations in metabolic levels. This study aimed to explore the metabolic differences between MDCK cells with varying proliferative capabilities through the use of both untargeted and targeted metabolomics. To investigate the metabolic discrepancies between adherent cell groups (MDCK-M60 and MDCK-CL23) and suspension cell groups (MDCK-XF04 and MDCK-XF06), untargeted and targeted metabolomics were used. Utilizing RT-qPCR analysis, the mRNA expressions of key metabolites enzymes were identified. An untargeted metabolomics study demonstrated the presence of 81 metabolites between MDCK-M60 and MDCK-CL23 cells, which were mainly affected by six pathways. An analysis of MDCK-XF04 and MDCK-XF06 cells revealed a total of 113 potential metabolites, the majority of which were impacted by ten pathways. Targeted metabolomics revealed a decrease in the levels of choline, tryptophan, and tyrosine in MDCK-CL23 cells, which was in accordance with the results of untargeted metabolomics. Additionally, MDCK-XF06 cells experienced a decrease in 5'-methylthioadenosine and tryptophan, while S-adenosylhomocysteine, kynurenine, 11Z-eicosenoic acid, 3-phosphoglycerate, glucose 6-phosphate, and phosphoenolpyruvic acid concentrations were increased. The mRNA levels of MAT1A, MAT2B, IDO1, and IDO2 in the two cell groups were all increased, suggesting that S-adenosylmethionine and tryptophan may have a significant role in cell metabolism. This research examines the effect of metabolite fluctuations on cell proliferation, thus offering a potential way to improve the rate of MDCK cell growth.

Sequential Activations of ChREBP and SREBP1 Signals Regulate the High-Carbohydrate Diet-Induced Hepatic Lipid Deposition in Gibel Carp (Carassius gibelio).

The present study investigated the sequential regulation signals of high-carbohydrate diet (HCD)-induced hepatic lipid deposition in gibel carp (Carassius gibelio). Two isonitrogenous and isolipidic diets, containing 25% (normal carbohydrate diet, NCD) and 45% (HCD) corn starch, were formulated to feed gibel carp (14.82 ± 0.04 g) for 8 weeks. The experimental fish were sampled at 2nd, 4th, 6th, and 8th week. In HCD group, the hyperlipidemia and significant hepatic lipid deposition (oil red O area and triglyceride content) was found at 4th, 6th, and 8th week, while the significant hyperglycemia was found at 2nd, 4th, and 8th week, compared to NCD group (P < 0.05). HCD induced hepatic lipid deposition via increased hepatic lipogenesis (acc, fasn, and acly) but not decreased hepatic lipolysis (hsl and cpt1a). When compared with NCD group, HCD significantly elevated the hepatic sterol regulatory element binding proteins 1 (SREBP1) signals (positive hepatocytes and fluorescence intensity) at 4th, 6th, and 8th week (P < 0.05). The hepatic SREBP1 signals increased from 2nd to 6th week, but decreased at 8th week due to substantiated insulin resistance (plasma insulin levels, plasma glucose levels, and P-AKTSer473 levels) in HCD group. Importantly, the hepatic carbohydrate response element binding protein (ChREBP) signals (positive hepatocytes, fluorescence intensity, and expression levels) were all significantly elevated by HCD-induced glucose-6-phosphate (G6P) accumulation at 2nd, 4th, 6th, and 8th week (P < 0.05). Compared to 2nd and 4th week, the hepatic ChREBP signals and G6P contents was significantly increased by HCD at 6th and 8th week (P < 0.05). The HCD-induced G6P accumulation was caused by the significantly increased expression of hepatic gck, pklr, and glut2 (P < 0.05) but not 6pfk at 4th, 6th, and 8th week, compared to NCD group. These results suggested that the HCD-induced hepatic lipid deposition was mainly promoted by SREBP1 in earlier stage and by ChREBP in later stage for gibel carp. This study revealed the sequential regulation pathways of the conversion from feed carbohydrate to body lipid in fish.

Abscisic Acid Induces Adventitious Rooting in Cucumber (Cucumis sativus L.) by Enhancing Sugar Synthesis.

Abscisic acid (ABA) affects many important plant processes, such as seed germination, root elongation and stomatal movement. However, little information is available about the relationship between ABA and sugar synthesis during adventitious root formation. The aim of this study was to evaluate the effect of ABA on adventitious root formation in cucumber and whether the effect of this plant hormone on sugar synthesis could be included as a causative factor for adventitious root development. We determined the contents of glucose, sucrose, starch, total sugar and sugar-related enzymes, including sucrose synthase (SS), sucrose phosphate synthase (SPS), hexokinase (HK) and pyruvate kinase (PK) activities in ABA treatment. We also quantified the relative expression of sucrose or glucose synthesis genes during this process. Increasing ABA concentrations significantly improved adventitious root formation, with the most considerable effect at 0.05 μM. Compared to the control, ABA treatment showed higher glucose, sucrose, starch and total sugar contents. Moreover, ABA treatment increased glucose-6-phosphate (G6P), fructose-6-phosphate (F6P) and glucose-1-phosphate (G1P) contents in cucumber explants during adventitious root development, which was followed by an increase of activities of sucrose-related enzymes SS and SPS, glucose-related enzymes HK and PK. ABA, meanwhile, upregulated the expression levels of sucrose or glucose synthesis-related genes, including CsSuSy1, CsSuSy6, CsHK1 and CsHK3. These results suggest that ABA may promote adventitious root development by increasing the contents of glucose, sucrose, starch, total sugar, G6P, F6P and G1P, the activities of SS, SPS, HK, SPS and the expression levels of CsSuSy1, CsSuSy6, CsHK1 and CsHK3 genes. These findings provide evidence for the physiological role of ABA during adventitious root formation and provide a new understanding of the possible relationship between ABA and sugar synthesis during adventitious rooting in plants.

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.

Kinetic Efficiency of R252L and D60N Mutants of C. elegans Glucose‐6‐Phosphate Dehydrogenase Relative to the Wild Type

Glucose-6-phosphate dehydrogenase (G6PD) is an enzyme present in all species that have thus far been tested (Vuillamy et al., 1988). G6PD is the rate limiting enzyme in the pentose phosphate pathway (PPP) (Wang and Engel, 2009) and reduces NADP+ to NADPH through the oxidation of glucose-6-phosphate (G6P) to 6-phospho-gluconolactone (Hirono et al. 1989). The effectiveness of the G6PD enzyme is vital to the health and survival of an organism, as the PPP is one of the major NADPH production mechanisms, and NADPH protects cells from succumbing to oxidative stress (Wang and Engel, 2009). In the present study, kinetic characterization of the C. elegans orthologs of two known deficient human G6PD variants was performed. Production of the R252L and D60N C. elegans G6PD variants was done via E. coli plasmid expression. Purification of the mutant enzymes was performed on a nickel column, and enzyme kinetics parameters of the mutant G6PD enzymes were determined and compared to those for the wild type enzyme. From the Michaelis-Menten kinetics assay results, it was determined that the Vmax value for the R252L mutant enzyme was 5.9 * 10-3 ± 7.5 * 10-4 µmol/min, the KM value was 1050 ± 200 µM, and the specific activity was 1.2 µmol/min/mg. For the D60N enzyme variant it was determined that the Vmax value was 3.2 * 10-3 ± 2.1 * 10 -4 µmol/min, the KM value was 80 ± 22 µM, and the specific activity was 6.5 µmol/min/mg. Lastly, the Vmax value for the wild type enzyme was 2.2 * 10-3 ± 1.4 * 10 -4 µmol/min, the KM value was 100 ± 27 µM, and the specific activity was 11.1 µmol/min/mg. These results diverge slightly from those previously published on the human orthologs of the two mutants. A 2013 study by Bendaoud et al. in which the human ortholog of the R252L GSPD mutant was first cited, as G6PD Tunisia, found that the mutant retained an estimated 25% of normal enzymatic activity while this study found that the R252L mutant had about 11% activity compared to the wild type. A 1988 study by Vuillamy et al. found that the Metaponto variant (the human ortholog of the D60N mutant) had a KM of 47 µM relative to G6P concentration, which is a little over half that observed in this study. The same study found that the enzymatic activity of the mutant within red blood cells was between 14 and 39% of the wild type while the present study found the activity of the D60N mutant to be about 59% of the wild type. This novel data presents evidence of the deficiency of the C. elegans orthologs of the Tunisia and Metaponto variants of human G6PD, thus illustrating their promise as model systems for their human counterparts. Bendaoud B, Hosni I, Mosbahi I, Hafsia R, Prehu C, Abbes S. 2013. Pathol Biol (Paris). 61(2):64–69. Hirono A, Kuhl W, Gelbart T, Forman L, Fairbanks VF, Beutler E. 1989. Proc Natl Acad Sci USA. 86(24):10015–10017. Vulliamy TJ, D'Urso M, Battistuzzi G, Estrada M, Foulkes NS, Martini G, Calabro V, Poggi V, Giordano R, Town M. 1988. Proc Natl Acad Sci U S A. 85(14):5171–5175. Wang X-T, Engel PC. 2009. Biochim Biophys Acta. 1792(8):804–809.

Detection of Low-Level Fosfomycin-Resistant Variants by Decreasing Glucose-6-Phosphate Concentration in Fosfomycin Susceptibility Determination.

Mutations that confer low-level fosfomycin resistance (LLFR) but not clinical resistance in Escherichia coli are increasingly reported. LLFR strains can become clinically resistant under urinary tract physiological conditions or may act as gateways for highly resistant subpopulations by the selection of additional LLFR mutations. Nevertheless, most LLFR strains are impossible to detect under routine fosfomycin susceptibility determinations. Here, we have explored the possibility of detecting LLFR variants by reducing glucose-6-phosphate (G6P) concentration in fosfomycin susceptibility testing for E. coli strains. As a proof of concept, fosfomycin minimal inhibitory concentrations (MICs) and disk diffusion susceptibility tests were performed for E. coli strain BW25113 and 10 isogenic derivatives carrying the most prevalent LLFR chromosomal mutations (∆uhpT, ∆glpT, ∆cyaA, and ∆ptsI) and their double combinations. Whereas standard G6P concentrations detected only ∆uhpT single and double variants, assays with reduced G6P detected all LLFR variants. In addition, G6P levels were determined to be ≤5 µg/mL in urine samples from 30 patients with urinary tract infection (UTI) caused by E. coli and 10 healthy volunteers, suggesting that most bacterial cells in uncomplicated UTIs are facing fosfomycin under low G6P concentration. Reducing G6P allows for the detection of LLFR variants, which may suppose a risk for future resistance development, especially in UTIs.

The absence of hepatic glucose-6 phosphatase/ChREBP couple is incompatible with survival in mice

ObjectiveGlucose production in the blood requires the expression of glucose-6 phosphatase (G6Pase), a key enzyme that allows glucose-6 phosphate (G6P) hydrolysis into free glucose and inorganic phosphate. We previously reported that the hepatic suppression of G6Pase leads to G6P accumulation and to metabolic reprogramming in hepatocytes from liver G6Pase-deficient mice (L.G6pc−/−). Interestingly, the activity of the transcription factor carbohydrate response element-binding protein (ChREBP), central for de novo lipid synthesis, is markedly activated in L.G6pc−/− mice, which consequently rapidly develop NAFLD-like pathology. In the current work, we assessed whether a selective deletion of ChREBP could prevent hepatic lipid accumulation and NAFLD initiation in L.G6pc−/− mice. MethodsWe generated liver-specific ChREBP (L.Chrebp−/−)- and/or G6Pase (L.G6pc−/−)-deficient mice using a Cre-lox strategy in B6.SACreERT2 mice. Mice were fed a standard chow diet or a high-fat diet for 10 days. Markers of hepatic metabolism and cellular stress were analysed in the liver of control, L. G6pc−/−, L. Chrebp−/− and double knockout (i.e., L.G6pc−/−.Chrebp−/−) mice. ResultsWe observed that there was a dramatic decrease in lipid accumulation in the liver of L.G6pc−/−.Chrebp−/− mice. At the mechanistic level, elevated G6P concentrations caused by lack of G6Pase are rerouted towards glycogen synthesis. Importantly, this exacerbated glycogen accumulation, leading to hepatic water retention and aggravated hepatomegaly. This caused animal distress and hepatocyte damage, characterised by ballooning and moderate fibrosis, paralleled with acute endoplasmic reticulum stress. ConclusionsOur study reveals the crucial role of the ChREBP-G6Pase duo in the regulation of G6P-regulated pathways in the liver.

Dehydration during storage affects carbohydrate metabolism and the accumulation of non-sucrose carbohydrates in postharvest sugarbeet roots

Sugarbeet roots produced in cold climates are stored in large piles prior to processing and are cooled by ambient winter air. These storage conditions dehydrate roots, causing root respiration rate and sucrose loss in storage to increase. Dehydration likely alters carbohydrate metabolism due to a greater demand for respiratory substrates and may affect the accumulation of non-sucrose carbohydrates that impede processing. However, effects of dehydration on carbohydrate metabolism of postharvest sugarbeet roots have not been studied. Research, therefore, was conducted to determine the effect of mild and severe dehydration on concentrations of the non-sucrose carbohydrates, glucose, fructose, and raffinose, their biosynthetic precursors, and the concentrations of metabolites that are intermediates in respiratory sucrose catabolism through glycolysis and the TCA cycle. Roots were stored at 85 and 40% relative humidity for up to 28 d to generate mildly and severely dehydrated roots typical of roots in the interior and exterior of storage piles. Severe dehydration was associated with reductions in raffinose, galactinol, glucose 6-phosphate, fructose 6-phosphate, phosphoenolpyruvate, and pyruvate concentrations, and increases in inositol and malate concentrations, while mild dehydration was associated with increased raffinose concentration. Concentrations of other glycolytic and TCA cycle intermediates, as well as the invert sugars, glucose and fructose, were largely unaffected by dehydration. From these results, it is concluded that raffinose accumulation and biosynthesis is highly sensitive to root dehydration and that carbohydrate metabolism through glycolysis is altered by dehydration, presumably to provide increased substrates for respiration. Overall, this research provides new insight into the impact of environmental conditions within storage piles on raffinose accumulation and the mechanisms by which dehydration accelerates sucrose loss in stored sugarbeet roots. • Raffinose accumulates in roots stored under mildly dehydrating conditions. • Raffinose content declines in roots stored under severely dehydrating conditions. • Severe dehydration alters flux through glycolysis to support elevated respiration. • Invert sugar concentration is not significantly altered by dehydration in storage.

Metabolic and Proteomic Perspectives of Augmentation of Nutritional Contents and Plant Defense in Vigna unguiculata

The current study enlists metabolites of Alstonia scholaris with bioactivities, and the most active compound, 3-(1-methylpyrrolidin-2-yl) pyridine, was selected against Macrophomina phaseolina. Appraisal of the Alstonia metabolites identified the 3-(1-methylpyrrolidin-2-yl) pyridine as a bioactive compound which elevated vitamins and nutritional contents of Vigna unguiculata up to ≥18%, and other physiological parameters up to 28.9%. The bioactive compound (0.1%) upregulated key defense genes, shifted defense metabolism from salicylic acid to jasmonic acid, and induced glucanase enzymes for improved defenses. The structural studies categorized four glucanase-isozymes under beta-glycanases falling in (Trans) glycosidases with TIM beta/alpha-barrel fold. The study determined key-protein factors (Q9SAJ4) for elevated nutritional contents, along with its structural and functional mechanisms, as well as interactions with other loci. The nicotine-docked Q9SAJ4 protein showed a 200% elevated activity and interacted with AT1G79550.2, AT1G12900.1, AT1G13440.1, AT3G04120.1, and AT3G26650.1 loci to ramp up the metabolic processes. Furthermore, the study emphasizes the physiological mechanism involved in the enrichment of the nutritional contents of V. unguiculata. Metabolic studies concluded that increased melibiose and glucose 6-phosphate contents, accompanied by reduced trehalose (-0.9-fold), with sugar drifts to downstream pyruvate biosynthesis and acetyl Co-A metabolism mainly triggered nutritional contents. Hydrogen bonding at residues G.357, G.380, and G.381 docked nicotine with Q9SAJ4 and transformed its bilobed structure for easy exposure toward substrate molecules. The current study augments the nutritional value of edible stuff and supports agriculture-based country economies.

Glucose 6-Phosphate Accumulates via Phosphoglucose Isomerase Inhibition in Heart Muscle.

Metabolic and structural remodeling is a hallmark of heart failure. This remodeling involves activation of the mTOR (mammalian target of rapamycin) signaling pathway, but little is known on how intermediary metabolites are integrated as metabolic signals. We investigated the metabolic control of cardiac glycolysis and explored the potential of glucose 6-phosphate (G6P) to regulate glycolytic flux and mTOR activation. We developed a kinetic model of cardiomyocyte carbohydrate metabolism, CardioGlyco, to study the metabolic control of myocardial glycolysis and G6P levels. Metabolic control analysis revealed that G6P concentration is dependent on phosphoglucose isomerase (PGI) activity. Next, we integrated ex vivo tracer studies with mathematical simulations to test how changes in glucose supply and glycolytic flux affect mTOR activation. Nutrient deprivation promoted a tight coupling between glucose uptake and oxidation, G6P reduction, and increased protein-protein interaction between hexokinase II and mTOR. We validated the in silico modeling in cultured adult mouse ventricular cardiomyocytes by modulating PGI activity using erythrose 4-phosphate. Inhibition of glycolytic flux at the level of PGI caused G6P accumulation, which correlated with increased mTOR activation. Using click chemistry, we labeled newly synthesized proteins and confirmed that inhibition of PGI increases protein synthesis. The reduction of PGI activity directly affects myocyte growth by regulating mTOR activation.

Diurnal biomarkers reveal key photosynthetic genes associated with increased oil palm yield.

To investigate limiters of photosynthate assimilation in the carbon-source limited crop, oil palm (Elaeis guineensis Jacq.), we measured differential metabolite, gene expression and the gas exchange in leaves in an open field for palms with distinct mesocarp oil content. We observed higher concentrations of glucose 1-phosphate, glucose 6-phosphate, sucrose 6-phosphate, and sucrose in high-oil content palms with the greatest difference being at 11:00 (p-value ≤0.05) immediately after the period of low morning light intensity. Three important photosynthetic genes were identified using differentially expressed gene analysis (DEGs) and were found to be significantly enriched through Gene Ontology (GO) and pathway enrichment: chlorophyll a-b binding protein (CAB-13), photosystem I (PSI), and Ferredoxin-NADP reductase (FNR), particularly for sampling points at non-peak light (11:00 and 19:00), ranging from 3.3-fold (PSI) and 5.6-fold (FNR) to 10.3-fold (CAB-13). Subsequent gas exchange measurements further supported increased carbon assimilation through higher level of internal CO2 concentration (Ci), stomatal conductance (gs) and transpiration rate (E) in high-oil content palms. The selection for higher expression of key photosynthesis genes together with CO2 assimilation under low light is likely to be important for crop improvement, in particular at full maturity and under high density planting regimes where light competition exists between palms.

Development of Voltammetric Glucose-6-phosphate Biosensors Based on the Immobilization of Glucose-6-phosphate Dehydrogenase on Polypyrrole- and Chitosan-Coated Fe3O4 Nanoparticles/Polypyrrole Nanocomposite Films.

Polypyrrole (PPy) and PPy-containing chitosan-coated Fe3O4 have been electrochemically polymerized on pencil graphite electrodes (PGEs). After the resulting electrodes were characterized by SEM-EDS analysis, glucose-6-phosphate dehydrogenase (G6PD) was immobilized onto these electrodes via glutaraldehyde. The biosensors prepared for the chronopotentiometric detection of glucose-6-phosphate (G6P) at 0.25 mAcm-2 were studied and optimized at different parameters such as the pH of supporting electrolyte, the temperature, and NADP+ and G6P concentrations related with the analytical performance of the biosensors. PPy/G6PD (BS-1) and CS/Fe3O4-PPy/G6PD (BS-2) biosensors showed a broad linear response in the concentration range 0.025-0.25mM and 0.0025-0.05mM, and their detection limits for G6P and the RSD values were determined as 0.008mM and 0.002mM and 3.80% and 4.60% after 15 times usage, respectively. The interference study with various major blood components such as urea, glucose, and cysteine was performed to evaluate the selectivity of the biosensors. The proposed BS-2 biosensor showed almost free response from available interferences in blood serum with a recovery of 91 to 110%. The developed biosensors could be used in the G6P level measurement of medical samples.

Metabolomic analysis of uterine serous carcinoma with acquired resistance to paclitaxel.

IntroductionUterine serous carcinoma (USC) is more aggressive than other subtypes of endometrial carcinoma and is associated with a poor prognosis. We analyzed the metabolomic profile of USC with acquired resistance to paclitaxel.ResultsGlutathione (GSH) concentration in PTX-1 cells was higher than in USPC-1 cells. In addition, GSH concentration in the USPC-1 cells increased after treatment with paclitaxel but was unchanged in PTX-1 cells. Glucose-6-phosphate (G6P) and ribose-5-phosphate (R5P) concentrations in PTX-1 cells were higher than those in USPC-1 cells. G6P concentration in the USPC-1 cells was unchanged after treatment with paclitaxel, while it decreased in PTX-1 cells.ConclusionOur results indicate that increased GSH and glucose metabolism may be related to acquiring resistance to paclitaxel in USC and thus may be targets for anti-USC therapy.Materials and MethodsWe compared metabolic profiles and reactions to paclitaxel in both a wild type USC cell line (USPC-1) and PTX-1, a cell line derived from USPC-1 which acquired paclitaxel resistance, using a capillary electrophoresis CE-MS/MS system.

Abstract 383: Metabolomic analysis of uterine serous carcinoma with acquired resistance to paclitaxel

Abstract Introduction: Uterine serous carcinoma (USC) is more aggressive than other subtypes of endometrial carcinoma and is associated with a poor prognosis. We analyzed the metabolomic profile of USC with acquired resistance to paclitaxel. Method: We compared metabolic profiles and reactions to paclitaxel in both a wild-type USC cell line (USPC-1) and PTX-1, a cell line derived from USPC-1 that acquired paclitaxel resistance, using a capillary electrophoresis CE-MS/MS system. Results: Glutathione (GSH) concentration in PTX-1 cells was higher than in USPC-1 cells. In addition, GSH concentration in the USPC-1 cells increased after treatment with paclitaxel but was unchanged in PTX-1 cells. Glucose-6-phosphate (G6P) and ribose-5-phosphate (R5P) concentrations in PTX-1 cells were higher than those in USPC-1 cells. G6P concentration in the USPC-1 cells was unchanged after treatment with paclitaxel, while it decreased in PTX-1 cells. Conclusion: Our results indicate that increased GSH and glucose metabolism may be related to acquiring resistance to paclitaxel in USC and thus may be targets for anti-USC therapy. Citation Format: Manabu Seino, Tsuyoshi Ohta, Hirotsugu Sakaki, Takeshi Sudo, Satoru Nagase. Metabolomic analysis of uterine serous carcinoma with acquired resistance to paclitaxel [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 383.

Heme Deficiency Causes Impaired Glycogen Synthesis in Skeletal Muscle Leading to Insulin Resistance

In vertebrates, the initial step in heme biosynthesis is the production of 5-aminolevulinic acid (ALA) by ALA synthase (ALAS). The ALAS1 gene encodes a ubiquitously expressed isozyme. Mice heterozygous null for ALAS1 (A1+/-s) experience mitochondrial dysfunction in skeletal muscle, impaired glucose tolerance (IGT) and insulin resistance (IR) past 20-weeks of age (aged A1+/-s) 1). IGT/IR in aged A1+/-s was remedied by oral administration of ALA for 1-week1). By contrast, mitochondrial dysfunction required 6-weeks of ALA administration before improvements could be observed, indicating the phenotype of IGT/ IR is not due to mitochondrial dysfunction1). We found no remarkable change in expressions and phosphorylation of proteins related to insulin signalling such as Akt and GSK3β in skeletal muscle of aged A1+/-s1). Rather, abnormal ultrastructure of glycogen granules and increased glycogen contents were observed in skeletal muscle of aged A1+/-s unlike of less than 15-weeks-old A1+/-s, and those abnormalities of glycogen were reversed after ALA-treatment for 1-week as well as IGT/IR. The quantification of glycogen contents showed that insulin-induced glycogen synthesis was impaired in skeletal muscle of aged A1+/-s, and the reduced de novo glycogen synthesis was recovered in ALA-administrated aged A1+/-s. Glycogen synthase (GS) activities were constitutively activated even at the low concentration of glucose 6-phosphate (G6P) and not augmented at the high concentration of G6P in extracts from skeletal muscle of aged A1+/-s, suggesting that GS fails to respond to allosteric activation by G6P. While, GS activities were normally regulated in those of ALA-administered aged A1+/-s. Taken together, our data suggest that impaired de novo glycogen synthesis in skeletal muscle is relevant to IGT/IR in aged A1+/-s due to constitutively activated GS without allosteric activation by G6P. Ref. 1) Saitoh et al. PLOS ONE in press. Disclosure O. Nakajima: None. S. Saitoh: Research Support; Spouse/Partner; Japan Society for the Promotion of Science, SBI Pharmaceuticals Co.,Ltd.. T. Kimura: None. T. Osaki: None. K.P. Vincent: Research Support; Self; Kora Healthcare. K. Takahashi: None. T. Tanaka: None. M. Nakajima: None.

Metabolomics reveals critical adrenergic regulatory checkpoints in glycolysis and pentose–phosphate pathways in embryonic heart

Cardiac energy demands during early embryonic periods are sufficiently met through glycolysis, but as development proceeds, the oxidative phosphorylation in mitochondria becomes increasingly vital. Adrenergic hormones are known to stimulate metabolism in adult mammals and are essential for embryonic development, but relatively little is known about their effects on metabolism in the embryonic heart. Here, we show that embryos lacking adrenergic stimulation have ∼10-fold less cardiac ATP compared with littermate controls. Despite this deficit in steady-state ATP, neither the rates of ATP formation nor degradation was affected in adrenergic hormone-deficient hearts, suggesting that ATP synthesis and hydrolysis mechanisms were fully operational. We thus hypothesized that adrenergic hormones stimulate metabolism of glucose to provide chemical substrates for oxidation in mitochondria. To test this hypothesis, we employed a metabolomics-based approach using LC/MS. Our results showed glucose 1-phosphate and glucose 6-phosphate concentrations were not significantly altered, but several downstream metabolites in both glycolytic and pentose-phosphate pathways were significantly lower compared with controls. Furthermore, we identified glyceraldehyde-3-phosphate dehydrogenase and glucose-6-phosphate dehydrogenase as key enzymes in those respective metabolic pathways whose activity was significantly (p < 0.05) and substantially (80 and 40%, respectively) lower in adrenergic hormone-deficient hearts. Addition of pyruvate and to a lesser extent ribose led to significant recovery of steady-state ATP concentrations. These results demonstrate that without adrenergic stimulation, glucose metabolism in the embryonic heart is severely impaired in multiple pathways, ultimately leading to insufficient metabolic substrate availability for successful transition to aerobic respiration needed for survival.

Species specificity of an adrenal androgen-mediated kill-switch triggered by p53 inactivation

Glucose-6-phosphate dehydrogenase (G6PD) is an oncoprotein that is regulated by the p53 tumor suppressor. Mutant p53 loses the ability to inhibit G6PD, and loss of G6PD control clearly plays a role in oncogenesis. The steroid hormone precursor dehydroepiandrosterone (DHEA) is an endogenous uncompetitive inhibitor of G6PD. In humans, and a few other species, the sulfated circulatory form of DHEA (DHEAS) is present at extremely high concentrations – much higher than can be accounted for by DHEA’s function as a precursor to steroid hormones. Uncompetitive inhibition is extremely rare in natural systems because it is irreversible in the presence of high concentrations of substrate and inhibitor. What has gone unappreciated is that such uncompetitive inhibition can quickly lead to cell death when the target is an obligatory housekeeping gene such as G6PD. Cells with inactivated p53 not only lose control over G6PD, but also over hexokinase (HK), the enzyme that converts glucose into glucose-6-phosphate (G6P), the substrate of G6PD. Furthermore, loss of p53 function de-represses NFκB activity, resulting in the upregulation of steroid sulfatase (SS) which converts circulating DHEAS into active DHEA. We propose that inactivation of p53 rapidly elevates G6P and DHEA concentrations in affected cells, driving uncompetitive inhibition of G6PD to lethal irreversibility. In animals with circulating DHEAS, this kill-switch mechanism may prevent most cases of p53 inactivation from becoming tumorigenic. Tumors would thus represent instances in which this mechanism had not been triggered, but which might still be triggered by application of DHEA sufficient to uncompetitively inhibit tumor G6PD. To test this hypothesis, we performed a pilot study in which dogs with cardiac hemangiosarcoma were treated with high dose (HD) DHEA supplemented with isoprene precursors to maintain geranylation of Rac GTPase. Tumor regression and longevity observed in these dogs supported the concept that some tumors retain extraordinary sensitivity to uncompetitive inhibition by DHEA.

Cortisol promotes endoplasmic glucose production via pyridine nucleotide redox.

Both increased adrenal and peripheral cortisol production, the latter governed by 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), contribute to the maintenance of fasting blood glucose. In the endoplasmic reticulum (ER), the pyridine nucleotide redox state (NADP/NADPH) is dictated by the concentration of glucose-6-phosphate (G6P) and the coordinated activities of two enzymes, hexose-6-phosphate dehydrogenase (H6PDH) and 11β-HSD1. However, luminal G6P may similarly serve as a substrate for hepatic glucose-6-phophatase (G6Pase). A tacit belief is that the G6P pool in the ER is equally accessible to both H6PDH and G6Pase. Based on our inhibition studies and kinetic analysis in isolated rat liver microsomes, these two aforesaid luminal enzymes do share the G6P pool in the ER, but not equally. Based on the kinetic modeling of G6P flux, the ER transporter for G6P (T1) preferentially delivers this substrate to G6Pase; hence, the luminal enzymes do not share G6P equally. Moreover, cortisol, acting through 11β-HSD1, begets a more reduced pyridine redox ratio. By altering this luminal redox ratio, G6P flux through H6PDH is restrained, allowing more G6P for the competing enzyme G6Pase. And, at low G6P concentrations in the ER lumen, which occur during fasting, this acute cortisol-induced redox adjustment promotes glucose production. This reproducible cortisol-driven mechanism has been heretofore unrecognized.