scholarly journals Measurement of pentose phosphate-pathway activity in a single incubation with [1,6-13C2,6,6-2H2]glucose

1994 ◽  
Vol 302 (1) ◽  
pp. 31-38 ◽  
Author(s):  
B D Ross ◽  
P B Kingsley ◽  
O Ben-Yoseph
Keyword(s):  
Pentose Phosphate Pathway ◽  
Cultured Cells ◽  
Krebs Cycle ◽  
Pentose Phosphate ◽  
Repeated Measurements ◽  
Co2 Production ◽  
Spectrometric Analysis ◽  
14Co2 Production ◽  
Single Set

The isotopically substituted molecule D-[1,6-13C2,6,6-2H2]glucose is introduced for measuring the relative activities of the pentose phosphate pathway (PPP) and glycolysis in a single incubation. PPP activity in cultured cells was determined by gas chromatography/mass spectrometric analysis of lactate produced by cells incubated with [1,6-13C2,6,6-2H2]glucose. Two other isotopes, [1,5,6-13C3]glucose and [6-13C,1,6,6-2H3]glucose, were not satisfactory for measurements of this activity. This method has four advantages over the traditional one in which 14CO2 production from [1-14C]glucose and [6-14C]glucose is compared: (1) repeated measurements can be made on a single set of cells, (2) only a single incubation is required, (3) extensive CO2 production by Krebs-cycle activity does not interfere with the measurements and (4) it is not necessary to measure the amount of glucose consumed in order to calculate relative activities of the PPP and glycolysis. Preliminary observation indicates that rat brain PPP activity may be measured in vivo with [1,6-13C2,6,6-2H2]glucose when combined with microdialysis techniques.

scholarly journals ELAPOR1 is a secretory granule maturation-promoting factor that is lost during paligenosis

2021 ◽  
Author(s):  
Charles J. Cho ◽  
Dongkook Park ◽  
Jason C. Mills
Keyword(s):  
Transcription Factor ◽  
Secretory Granule ◽  
Cultured Cells ◽  
Tissue Injury ◽  
Pancreatic Acinar Cells ◽  
Secretory Cells ◽  
Spectrometric Analysis ◽  
Granule Maturation

A single transcription factor, MIST1 (BHLHA15), maximizes secretory function in diverse secretory cells (like pancreatic acinar cells) by transcriptionally upregulating genes that elaborate secretory architecture. Here, we show that the scantly-studied MIST1 target, ELAPOR1, is an evolutionarily conserved, novel Mannose-6-phosphate receptor (M6PR) domain-containing protein. ELAPOR1 expression was specific to zymogenic cells (ZCs, the MIST1-expressing population in the stomach). ELAPOR1 expression was lost as tissue injury caused ZCs to undergo paligenosis (ie, to become metaplastic and reenter the cell cycle). In cultured cells, ELAPOR1 trafficked with cis-Golgi resident proteins and with the trans-Golgi and late endosome protein: cation-independent M6PR. Secretory vesicle trafficking was disrupted by expression of ELAPOR1 truncation mutants. Mass spectrometric analysis of co-immunoprecipitated proteins showed ELAPOR1 and CI-M6PR shared many binding partners. However, CI-M6PR and ELAPOR1 must function differently, as CI-M6PR co-immunoprecipitated more lysosomal proteins and was not decreased during paligenosis in vivo. We generated Elapor1−/− mice to determine ELAPOR1 function in vivo. Consistent with in vitro findings, secretory granule maturation was defective in Elapor1−/− ZCs. Our results identify a role for ELAPOR1 in secretory granule maturation and help clarify how a single transcription factor maintains mature exocrine cell architecture in homeostasis and helps dismantles it during paligenosis.

scholarly journals YvcK of Bacillus subtilis is required for a normal cell shape and for growth on Krebs cycle intermediates and substrates of the pentose phosphate pathway

Microbiology ◽  
2005 ◽  
Vol 151 (11) ◽  
pp. 3777-3791 ◽  
Author(s):  
Boris Görke ◽  
Elodie Foulquier ◽  
Anne Galinier
Keyword(s):  
Bacillus Subtilis ◽  
Pentose Phosphate Pathway ◽  
Carbon Metabolism ◽  
Carbon Sources ◽  
Krebs Cycle ◽  
Normal Growth ◽  
Central Carbon Metabolism ◽  
Pentose Phosphate ◽  
Homologous Protein ◽  
Krebs Cycle Intermediates

The HPr-like protein Crh has so far been detected only in the bacillus group of bacteria. In Bacillus subtilis, its gene is part of an operon composed of six ORFs, three of which exhibit strong similarity to genes of unknown function present in many bacteria. The promoter of the operon was determined and found to be constitutively active. A deletion analysis revealed that gene yvcK, encoded by this operon, is essential for growth on Krebs cycle intermediates and on carbon sources metabolized via the pentose phosphate pathway. In addition, cells lacking YvcK acquired media-dependent filamentous or L-shape-like aberrant morphologies. The presence of high magnesium concentrations restored normal growth and cell morphology. Furthermore, suppressor mutants cured from these growth defects appeared spontaneously with a high frequency. Such suppressing mutations were identified in a transposon mutagenesis screen and found to reside in seven different loci. Two of them mapped in genes of central carbon metabolism, including zwf, which encodes glucose-6-phosphate dehydrogenase and cggR, the product of which regulates the synthesis of glyceraldehyde-3-phosphate dehydrogenase. All these results suggest that YvcK has an important role in carbon metabolism, probably in gluconeogenesis required for the synthesis of cell wall precursor molecules. Interestingly, the Escherichia coli homologous protein, YbhK, can substitute for YvcK in B. subtilis, suggesting that the two proteins have been functionally conserved in these different bacteria.

scholarly journals CBMT-08. IN VIVO EVALUATION OF PENTOSE PHOSPHATE PATHWAY ACTIVITY IN ORTHOTOPIC GLIOMA USING HYPERPOLARIZED δ-[1-13C]GLUCONOLACTONE

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi34-vi34
Author(s):  
Georgios Batsios ◽  
Pavithra Viswanath ◽  
Peng Cao ◽  
Celine Taglang ◽  
Elavarasan Subramani ◽  
...  
Keyword(s):  
Pentose Phosphate Pathway ◽  
Tumor Development ◽  
Pentose Phosphate ◽  
Response To Therapy ◽  
Metabolic Imaging ◽  
Normal Brain ◽  
In Vivo Evaluation ◽  
First Time ◽  
Orthotopic Glioma

Abstract The pentose phosphate pathway (PPP) generates NADPH and ribose 5-phosphate, which are involved in the scavenging of reactive oxygen species and the synthesis of nucleotides. As such, the PPP is typically upregulated in cancer cells to address the metabolic needs of rapid cell proliferation. Imaging PPP upregulation could therefore be useful in tumor assessment. One intermediate of the pathway is 6-phospho-δ-gluconolactone (6P-δ-GL), which is produced by phosphorylation of δ-gluconolactone. 6P-δ-GL is further metabolized to 6-phospho-gluconate (6PG). The goal of our study was to evaluate, for the first time, whether hyperpolarized (HP) δ-[1-13C]gluconolactone can be used to assess PPP flux and detect the presence of tumor in an orthotopic glioma rat model. Athymic nude rats bearing orthotropic U87 tumors or age-matched tumor-free controls were investigated. HP studies were performed following intravenous injection of HP δ-[1-13C]gluconolactone and metabolic images using a flyback spectral-spatial echo-planar spectroscopic imaging pulse were acquired. The data were processed using in-house Matlab code. 6P-δ-GL and 6-phospho-γ-[1-13C]gluconolactone were observed in all rats ~10 seconds after HP δ-[1-13C]gluconolactone injection, followed ~5 seconds later by production of 6PG observed at 179.3ppm. These data indicate that HP δ-[1-13C]gluconolactone likely crosses the blood-brain barrier, consistent with its transport via glucose transporters, and is rapidly metabolized. Importantly, 6PG was significantly higher in tumor voxels. The ratio of 6PG-to-6P-δ-GL was comparable in normal brain and in normal-appearing contralateral brain of tumor-bearing rats at 0.43±0.09 and 0.45±0.06 respectively (p=0.85), but significant higher in the tumor regions at 0.70±0.11 (p=0.04 and p=0.02 respectively), consistent with the elevated PPP flux that typically occurs in tumor cells. Our results indicate, to our knowledge for the first time, that metabolism of HP δ-[1-13C]gluconolactone can be assessed in the brain and that elevated 6PG production in glioma provides a potential metabolic imaging approach to probe tumor development, recurrence and response to therapy.

scholarly journals Beneficial effects of acute inhibition of the oxidative pentose phosphate pathway in the failing heart

2014 ◽  
Vol 306 (5) ◽  
pp. H709-H717 ◽  
Author(s):  
Claudio Vimercati ◽  
Khaled Qanud ◽  
Gianfranco Mitacchione ◽  
Danuta Sosnowska ◽  
Zoltan Ungvari ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Pentose Phosphate Pathway ◽  
Tissue Concentration ◽  
Glucose Consumption ◽  
Pentose Phosphate ◽  
Oxidative Pentose Phosphate Pathway ◽  
Stroke Work ◽  
Failing Heart

In vitro studies suggested that glucose metabolism through the oxidative pentose phosphate pathway (oxPPP) can paradoxically feed superoxide-generating enzymes in failing hearts. We therefore tested the hypothesis that acute inhibition of the oxPPP reduces oxidative stress and enhances function and metabolism of the failing heart, in vivo. In 10 chronically instrumented dogs, congestive heart failure (HF) was induced by high-frequency cardiac pacing. Myocardial glucose consumption was enhanced by raising arterial glycemia to levels mimicking postprandial peaks, before and after intravenous administration of the oxPPP inhibitor 6-aminonicotinamide (80 mg/kg). Myocardial energy substrate metabolism was measured with radiolabeled glucose and oleic acid, and cardiac 8-isoprostane output was used as an index of oxidative stress. A group of five chronically instrumented, normal dogs served as control. In HF, raising glycemic levels from ∼80 to ∼170 mg/dL increased cardiac isoprostane output by approximately twofold, whereas oxPPP inhibition normalized oxidative stress and enhanced cardiac oxygen consumption, glucose oxidation, and stroke work. In normal hearts glucose infusion did not induce significant changes in cardiac oxidative stress. Myocardial tissue concentration of 6P-gluconate, an intermediate metabolite of the oxPPP, was significantly reduced by ∼50% in treated versus nontreated failing hearts, supporting the inhibitory effect of 6-aminonicotinamide. Our study indicates an important contribution of the oxPPP activity to cardiac oxidative stress in HF, which is particularly pronounced during common physiological changes such as postprandial glycemic peaks.

scholarly journals Role of Intracellular Carbon Metabolism Pathways in Shigella flexneri Virulence

2014 ◽  
Vol 82 (7) ◽  
pp. 2746-2755 ◽  
Author(s):  
E. A. Waligora ◽  
C. R. Fisher ◽  
N. J. Hanovice ◽  
A. Rodou ◽  
E. E. Wyckoff ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Pentose Phosphate Pathway ◽  
Carbon Metabolism ◽  
Cultured Cells ◽  
Shigella Flexneri ◽  
Plaque Assay ◽  
Pentose Phosphate ◽  
Host Cells ◽  
Content Type ◽  
Pathway Gene

ABSTRACTShigella flexneri, which replicates in the cytoplasm of intestinal epithelial cells, can use the Embden-Meyerhof-Parnas, Entner-Doudoroff, or pentose phosphate pathway for glycolytic carbon metabolism. To determine which of these pathways is used by intracellularS. flexneri, mutants were constructed and tested in a plaque assay for the ability to invade, replicate intracellularly, and spread to adjacent epithelial cells. Mutants blocked in the Embden-Meyerhof-Parnas pathway (pfkABandpykAFmutants) invaded the cells but formed very small plaques. Loss of the Entner-Doudoroff pathway geneedaresulted in small plaques, but the doubleeda eddmutant formed normal-size plaques. This suggested that the plaque defect of theedamutant was due to buildup of the toxic intermediate 2-keto-3-deoxy-6-phosphogluconic acid rather than a specific requirement for this pathway. Loss of the pentose phosphate pathway had no effect on plaque formation, indicating that it is not critical for intracellularS. flexneri. Supplementation of the epithelial cell culture medium with pyruvate allowed the glycolysis mutants to form larger plaques than those observed with unsupplemented medium, consistent with data from phenotypic microarrays (Biolog) indicating that pyruvate metabolism was not disrupted in these mutants. Interestingly, the wild-typeS. flexnerialso formed larger plaques in the presence of supplemental pyruvate or glucose, with pyruvate yielding the largest plaques. Analysis of the metabolites in the cultured cells showed increased intracellular levels of the added compound. Pyruvate increased the growth rate ofS. flexneriin vitro, suggesting that it may be a preferred carbon source inside host cells.

A study of the relationship between seed dormancy and pentose phosphate pathway activity in Avena fatua

1983 ◽  
Vol 61 (3) ◽  
pp. 667-670 ◽  
Author(s):  
E. P. Fuerst ◽  
M. K. Upadhyaya ◽  
G. M. Simpson ◽  
J. M. Naylor ◽  
S. W. Adkins
Keyword(s):  
Seed Dormancy ◽  
Pentose Phosphate Pathway ◽  
Krebs Cycle ◽  
Pure Line ◽  
Pentose Phosphate ◽  
Avena Fatua ◽  
Wild Oats ◽  
Gibberellin Treatment ◽  
The Relationship ◽  
Increased Activity

The hypothesis that loss of seed dormancy is associated with an increased activity of the pentose phosphate pathway (PPP) relative to glycolysis and the Krebs cycle was tested. The PPP activity was monitored by measuring the C6/C1 ratio in embryos excised from incubated caryopses of two genetically pure nondormant (ND) lines and in three dormant (D) lines of Avena fatua L., the wild oat. The C6/C1 ratios of all lines were similar at the commencement of incubation. In the two ND lines the ratio increased steadily prior to and during emergence of the radicle. In the three D lines the ratio increased during the first 24 h and then remained almost constant; there was no germination. When gibberellin treatment was used to overcome dormancy in the D lines, the C6/C1 ratio increased during the first 24 h in two of the lines and continued to increase parallel to germination in a manner similar to normal germination in ND lines. In the third D line, despite loss of dormancy from gibberellin treatment, the ratio did not increase after 24 h. Loss of dormancy during dry storage of seeds of a D-type pure line was accompanied by an increase in the C6/C1 ratio, as measured in freshly imbibed seeds. This indicates a decreased activity of the PPP relative to glycolysis and the Krebs cycle. These findings are contrary to Roberts's hypothesis that loss of dormancy in wild oats is associated with a relative decrease in the C6/C1 ratio.

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