scholarly journals Quantitative analysis of intermediary metabolism in hepatocytes incubated in the presence and absence of glucagon with a substrate mixture containing glucose, ribose, fructose, alanine and acetate

1985 ◽  
Vol 225 (3) ◽  
pp. 761-786 ◽  
Author(s):  
M Rabkin ◽  
J J Blum
Keyword(s):  
Pyruvate Kinase ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Krebs Cycle ◽  
Metabolic Model ◽  
Pentose Phosphate ◽  
Atp Production ◽  
Considerable Excess ◽  
Net Flux ◽  
Presence And Absence ◽  
Pentose Phosphate Pathways

Hepatocytes were isolated from the livers of fed rats and incubated, in the presence and absence of 100 nM-glucagon, with a substrate mixture containing glucose (10 mM), fructose (4 mM), alanine (3.5 mM), acetate (1.25 mM), and ribose (1 mM). In any given incubation one substrate was labelled with 14C. Incorporation of 14C into glucose, glycogen, CO2, lactate, alanine, glutamate, lipid glycerol and fatty acids was measured after 20 and 40 min of incubation under quasi-steady-state conditions [Borowitz, Stein & Blum (1977) J. Biol. Chem. 252, 1589-1605]. These data and the measured O2 consumption were analysed with the aid of a structural metabolic model incorporating all reactions of the glycolytic, gluconeogenic, and pentose phosphate pathways, and associated mitochondrial and cytosolic reactions. A considerable excess of experimental measurements over independent flux parameters and a number of independent measurements of changes in metabolite concentrations allowed for a stringent test of the model. A satisfactory fit to the data was obtained for each condition. Significant findings included: control cells were glycogenic and glucagon-treated cells glycogenolytic during the second interval; an ordered (last in, first out) model of glycogen degradation [Devos & Hers (1979) Eur. J. Biochem. 99, 161-167] was required in order to fit the experimental data; the pentose shunt contributed approx. 15% of the carbon for gluconeogenesis in both control and glucagon-treated cells; net flux through the lower Embden-Meyerhof pathway was in the glycolytic direction except during the 20-40 min interval in glucagon-treated cells; the increased gluconeogenesis in response to glucagon was correlated with a decreased pyruvate kinase flux and lactate output; fluxes through pyruvate kinase, pyruvate carboxylase, and phosphoenolpyruvate carboxykinase were not coordinately controlled; Krebs cycle activity did not change with glucagon treatment; flux through the malic enzyme was towards pyruvate formation except for control cells during interval II; and ‘futile’ cycling at each of the five substrate cycles examined (including a previously undescribed cycle at acetate/acetyl-CoA) consumed about 26% of cellular ATP production in control hepatocytes and 21% in glucagon-treated cells.

scholarly journals Quantitative analysis of flux along the gluconeogenic, glycolytic and pentose phosphate pathways under reducing conditions in hepatocytes isolated from fed rats

1983 ◽  
Vol 212 (3) ◽  
pp. 585-598 ◽  
Author(s):  
J M Crawford ◽  
J J Blum
Keyword(s):  
Compartment Model ◽  
Pentose Phosphate ◽  
Improved Method ◽  
Single Compartment ◽  
Reducing Conditions ◽  
Single Compartment Model ◽  
Net Flux ◽  
Pentose Phosphate Pathways ◽  
Fructose 1,6 Bisphosphate ◽  
Hexose Phosphates

Hepatocytes were isolated from the livers of fed rats and incubated with a mixture of glucose (10 mM), ribose (1 mM), mannose (4 mM), glycerol (3 mM), acetate (1.25 mM), and ethanol (5 mM) with one substrate labelled with 14C in any given incubation. Incorporation of label into CO2, glucose, glycogen, lipid glycerol and fatty acids, acetate and C-1 of glucose was measured at 20 and 40 min after the start of the incubation. The data (about 48 measurements for each interval) were used in conjunction with a single-compartment model of the reactions of the gluconeogenic, glycolytic and pentose phosphate pathways and a simplified model of the relevant mitochondrial reactions. An improved method of computer analysis of the equations describing the flow of label through each carbon atom of each metabolite under steady-state conditions was used to compute values for the 34 independent flux parameters in this model. A good fit to the data was obtained, thereby permitting good estimates of most of the fluxes in the pathways under consideration. The data show that: net flux above the level of the triose phosphates is gluconeogenic; label in the hexose phosphates is fully equilibrated by the second 20 min interval; the triose phosphate isomerase step does not equilibrate label between the triose phosphates; substrate cycles are operating at the glucose-glucose 6-phosphate, fructose 6-phosphate-fructose 1,6-bisphosphate and phosphoenolpyruvate-pyruvate-oxaloacetate cycles; and, although net flux through the enzymes catalysing the non-oxidative steps of the pentose phosphate pathway is small, bidirectional fluxes are large.

scholarly journals Quantitative analysis of intermediary metabolism in rat hepatocytes incubated in the presence and absence of ethanol with a substrate mixture including ketoleucine

1989 ◽  
Vol 258 (1) ◽  
pp. 121-140 ◽  
Author(s):  
J M Baranyai ◽  
J J Blum
Keyword(s):  
Ketone Bodies ◽  
Rat Hepatocytes ◽  
Metabolic Model ◽  
Oxygen Utilization ◽  
Acetyl Coa ◽  
Atp Production ◽  
Considerable Excess ◽  
Metabolic Conditions ◽  
Label Incorporation

Hepatocytes isolated from livers of fed rats were incubated with a mixture of glucose (10 mM), ribose (1.0 mM), acetate (1.25 mM), alanine (3.5 mM), glutamate (2.0 mM), aspartate (2.0 mM), 4-methyl-2-oxovaleric acid (ketoleucine) (3.0 mM), and, in paired flasks, 10 mM-ethanol. One substrate was 14C-radiolabelled in any given incubation. Incorporation of 14C into glucose, glycogen, CO2, lactate, alanine, aspartate, glutamate, acetate, urea, lipid glycerol, fatty acids and the 1- and 2,3,4-positions of ketone bodies was measured after 20 and 40 min of incubation under quasi-steady-state conditions. Data were analysed with the aid of a realistic structural metabolic model. In each of the four conditions examined, there were approx. 77 label incorporation measurements and several measurements of changes in metabolite concentrations. The considerable excess of measurements over the 37 independent flux parameters allowed for a stringent test of the model. A satisfactory fit to these data was obtained for each condition. There were large bidirectional fluxes along the gluconeogenic/glycolytic pathways, with net gluconeogenesis. Rates of ureagenesis, oxygen consumption and ketogenesis were high under all four conditions studied. Oxygen utilization was accurately predicted by three of the four models. There was complete equilibration between mitochondrial and cytosolic pools of acetate and of CO2, but for several of the metabolic conditions, two incompletely equilibrated pools of mitochondrial acetyl-CoA and oxaloacetate were required. Ketoleucine was utilized at a rate comparable to that reported by others in perfused liver and entered the mitochondrial pool of acetyl-CoA directly associated with ketone body formation. Ethanol, which was metabolized at rates comparable to those in vivo, caused relatively few changes in overall flux patterns. Several effects related to the increased NADH/NAD+ ratio were observed. Pyruvate dehydrogenase was completely inhibited and the ratio of acetoacetate to 3-hydroxybutyrate was decreased; flux through glutamate dehydrogenase, the citric acid cycle, and ketoleucine dehydrogenase were, however, only slightly inhibited. Net production of ATP occurred in all conditions studied and was increased by ethanol. Futile cycling was quantified at the glucose/glucose 6-phosphate, glycogen/glucose 6-phosphate, fructose 6-phosphate/fructose 1,6-bis-phosphate, and phosphoenolpyruvate/pyruvate/oxaloacetate substrate cycles. Cycling at these four loci consumed about 22% of cellular ATP production in control hepatocytes and 14% in ethanol-treated cells.

Isoenzymes of the glycolytic and pentose-phosphate pathways during the development of the castor oil seed

1981 ◽  
Vol 59 (8) ◽  
pp. 1423-1425 ◽  
Author(s):  
Robert J. Ireland ◽  
David T. Dennis
Keyword(s):  
Pyruvate Kinase ◽  
Castor Oil ◽  
Ricinus Communis ◽  
Pentose Phosphate ◽  
Phosphogluconate Dehydrogenase ◽  
Hexose Phosphate ◽  
Oil Synthesis ◽  
Ricinus Communis L ◽  
Oil Plant ◽  
Pentose Phosphate Pathways

Plastid and cytosolic isozymes of pyruvate kinase, hexose-phosphate isomerase, and 6-phosphogluconate dehydrogenase have been isolated and separated from seeds of the castor oil plant (Ricinus communis L.) at different stages of development. These enzymes can be detected 24 days after pollination, when the plastids contain 14% of the total cellular activities of both the pyruvate kinase and hexose-phosphate isomerase and 40% of the 6-phosphogluconate dehydrogenase activities. At this stage the seed consists mainly of nucellus, that is subsequently replaced by endosperm. The activities of the plastid isozymes increase faster than the cytosolic isozymes, until after 40 days, when endosperm fills the seed, the plastids contain 25% of the hexose-phosphate isomerase, 70% of the 6-phosphogluconate dehydrogenase, and 40% of the pyruvate kinase activities. The development of the plastid isozymes correlates with the increase in oil synthesis. This supports a central role for plastids in oil synthesis.

Glyconeogenesis from lactate in frog striated muscle

1979 ◽  
Vol 237 (5) ◽  
pp. C231-C236 ◽  
Author(s):  
R. J. Connett
Keyword(s):  
Equilibrium Constant ◽  
Pyruvate Kinase ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Striated Muscle ◽  
Krebs Cycle ◽  
Mass Action ◽  
Acetate Incorporation ◽  
Krebs Cycle Intermediates ◽  
Frog Sartorius ◽  
Stimulation Of

The rates of 14carbon incorporation into CO2 and glycogen from [U-14C]-lactate and [1-14C]acetate in frog sartorius muscles were compared. The rates of incorporation into CO2 were similar, while the rate of incorporation of lactate into glycogen was more than 200-fold larger than that of acetate incorporation. It is concluded that the pathway of lactate incorporation into glycogen does not involve Krebs cycle intermediates and is extramitochondrial. To test the possibility that the pathway of lactate incorporation involves net reversal of a pyruvate kinase, the changes in phosphoenolpyruvate and pyruvate concentrations during stimulation of lactate incorporation into glycogen were measured. There wer none. The mass action ratio of pyruvate kinase was calculated. This value was two orders of magnitude from the equilibrium constant and it was concluded that reversal of pyruvate kinase was a very unlikely pathway. To test the possibility that a pathway involving the oxaloacetate-to-phosphoenolpyruvate step was involved the muscles were treated with 3-mercaptopicolinate, an inhibitor of phosphoenolpyruvate carboxykinase. The treatment resulted in decreased incorporation of lactate into glycogen.

scholarly journals Cinnamon Oil Inhibits Penicillium expansum Growth by Disturbing the Carbohydrate Metabolic Process

2021 ◽  
Vol 7 (2) ◽  
pp. 123
Author(s):  
Tongfei Lai ◽  
Yangying Sun ◽  
Yaoyao Liu ◽  
Ran Li ◽  
Yuanzhi Chen ◽  
...  
Keyword(s):  
Pentose Phosphate ◽  
Metabolic Process ◽  
Penicillium Expansum ◽  
Economic Losses ◽  
Pome Fruit ◽  
Cinnamon Oil ◽  
Atp Production ◽  
Protein Levels ◽  
Regulatory Enzymes ◽  
Expression Of Genes

Penicillium expansum is a major postharvest pathogen that mainly threatens the global pome fruit industry and causes great economic losses annually. In the present study, the antifungal effects and potential mechanism of cinnamon oil against P. expansum were investigated. Results indicated that 0.25 mg L−1 cinnamon oil could efficiently inhibit the spore germination, conidial production, mycelial accumulation, and expansion of P. expansum. In addition, it could effectively control blue mold rots induced by P. expansum in apples. Cinnamon oil could also reduce the expression of genes involved in patulin biosynthesis. Through a proteomic quantitative analysis, a total of 146 differentially expressed proteins (DEPs) involved in the carbohydrate metabolic process, most of which were down-regulated, were noticed for their large number and functional significance. Meanwhile, the expressions of 14 candidate genes corresponding to DEPs and the activities of six key regulatory enzymes (involving in cellulose hydrolyzation, Krebs circle, glycolysis, and pentose phosphate pathway) showed a similar trend in protein levels. In addition, extracellular carbohydrate consumption, intracellular carbohydrate accumulation, and ATP production of P. expansum under cinnamon oil stress were significantly decreased. Basing on the correlated and mutually authenticated results, we speculated that disturbing the fungal carbohydrate metabolic process would be partly responsible for the inhibitory effects of cinnamon oil on P. expansum growth. The findings would provide new insights into the antimicrobial mode of cinnamon oil.

scholarly journals Properties of pyruvate kinase and phosphoenolpyruvate carboxykinase in relation to the direction and regulation of phosphoenolpyruvate metabolism in muscles of the frog and marine invertebrates

1978 ◽  
Vol 174 (3) ◽  
pp. 979-987 ◽  
Author(s):  
Victor A. Zammit ◽  
Eric A. Newsholme
Keyword(s):  
Pyruvate Kinase ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Marine Invertebrates ◽  
Adductor Muscle ◽  
Sea Anemone ◽  
Anaerobic Conditions ◽  
Fructose Bisphosphate ◽  
Low Concentrations ◽  
Optimum Ph ◽  
Hill Coefficients

1. The properties of pyruvate kinase and, if present, phosphoenolpyruvate carboxykinase from the muscles of the sea anemone, scallop, oyster, crab, lobster and frog were investigated. 2. In general, the properties of pyruvate kinase from all muscles were similar, except for those of the enzyme from the oyster (adductor muscle); the pH optima were between 7.1 and 7.4, whereas that for oyster was 8.2; fructose bisphosphate lowered the optimum pH of the oyster enzyme from 8.2 to 7.1, but it had no effect on the enzymes from other muscles. Hill coefficients for the effect of the concentration of phosphoenolpyruvate were close to unity in the absence of added alanine for the enzymes from all muscles except oyster adductor muscle; it was 1.5 for this enzyme. Alanine inhibited the enzyme from all muscles except the frog; this inhibition was relieved by fructose bisphosphate. Low concentrations of alanine were very effective with the enzyme from the oyster (50% inhibition was observed at 0.4mm). Fructose bisphosphate activated the enzyme from all muscles, but extremely low concentrations were effective with the oyster enzyme (0.13μm produced 50% activation). 3. In general, the properties of phosphoenolpyruvate carboxykinase from the sea anemone and oyster muscles are similar: the Km values for phosphoenolpyruvate are low (0.10 and 0.13mm); the enzymes require Mn2+ in addition to Mg2+ for activity; and ITP inhibits the enzymes and the inhibition is relieved by alanine. These latter compounds had no effect on enzymes from other muscles. 4. It is suggested that changes in concentrations of fructose bisphosphate, alanine and ITP produce a coordinated mechanism of control of the activities of pyruvate kinase and phosphoenolpyruvate carboxykinase in the sea anemone and oyster muscles, which ensures that phosphoenolpyruvate is converted into oxaloacetate and then into succinate in these muscles under anaerobic conditions. 5. It is suggested that in the muscles of the crab, lobster and frog, phosphoenolpyruvate carboxykinase catalyses the conversion of oxaloacetate into phosphoenolpyruvate. This may be part of a pathway for the oxidation of some amino acids in these muscles.

scholarly journals Novel Antarctic yeast adapts to cold by switching energy metabolism and increasing small RNA synthesis

2021 ◽  
Author(s):  
D. Touchette ◽  
I. Altshuler ◽  
C. Gostinčar ◽  
P. Zalar ◽  
I. Raymond-Bouchard ◽  
...  
Keyword(s):  
Ethanol Production ◽  
Ethanol Fermentation ◽  
Phylogenetic Analyses ◽  
Regulation Of Gene Expression ◽  
Pentose Phosphate ◽  
Metabolic Switch ◽  
Antarctic Yeast ◽  
Fungal Adaptation ◽  
Pentose Phosphate Pathways ◽  
Post Transcriptional Regulation

AbstractThe novel extremophilic yeast Rhodotorula frigidialcoholis, formerly R. JG1b, was isolated from ice-cemented permafrost in University Valley (Antarctic), one of coldest and driest environments on Earth. Phenotypic and phylogenetic analyses classified R. frigidialcoholis as a novel species. To characterize its cold-adaptive strategies, we performed mRNA and sRNA transcriptomic analyses, phenotypic profiling, and assessed ethanol production at 0 and 23 °C. Downregulation of the ETC and citrate cycle genes, overexpression of fermentation and pentose phosphate pathways genes, growth without reduction of tetrazolium dye, and our discovery of ethanol production at 0 °C indicate that R. frigidialcoholis induces a metabolic switch from respiration to ethanol fermentation as adaptation in Antarctic permafrost. This is the first report of microbial ethanol fermentation utilized as the major energy pathway in response to cold and the coldest temperature reported for natural ethanol production. R. frigidialcoholis increased its diversity and abundance of sRNAs when grown at 0 versus 23 °C. This was consistent with increase in transcription of Dicer, a key protein for sRNA processing. Our results strongly imply that post-transcriptional regulation of gene expression and mRNA silencing may be a novel evolutionary fungal adaptation in the cryosphere.

scholarly journals Metabolome Shift in Both Metastatic Breast Cancer Cells and Astrocytes Which May Contribute to the Tumor Microenvironment

2021 ◽  
Vol 22 (14) ◽  
pp. 7430
Author(s):  
Hiromi Sato ◽  
Ayaka Shimizu ◽  
Toya Okawa ◽  
Miaki Uzu ◽  
Momoko Goto ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Tumor Microenvironment ◽  
Cancer Cells ◽  
Metastatic Breast ◽  
Principal Component ◽  
Pentose Phosphate ◽  
Metastatic Brain Tumors ◽  
Pentose Phosphate Pathways ◽  
Culture Supernatants

The role of astrocytes in the periphery of metastatic brain tumors is unclear. Since astrocytes regulate central nervous metabolism, we hypothesized that changes in astrocytes induced by contact with cancer cells would appear in the metabolome of both cells and contribute to malignant transformation. Coculture of astrocytes with breast cancer cell supernatants altered glutamate (Glu)-centered arginine–proline metabolism. Similarly, the metabolome of cancer cells was also altered by astrocyte culture supernatants, and the changes were further amplified in astrocytes exposed to Glu. Inhibition of Glu uptake in astrocytes reduces the variability in cancer cells. Principal component analysis of the cancer cells revealed that all these changes were in the first principal component (PC1) axis, where the responsible metabolites were involved in the metabolism of the arginine–proline, pyrimidine, and pentose phosphate pathways. The contribution of these changes to the tumor microenvironment needs to be further pursued.

scholarly journals Intracellular Mg2+regulates ADP phosphorylation and adenine nucleotide synthesis in human erythrocytes

1998 ◽  
Vol 274 (5) ◽  
pp. E920-E927 ◽  
Author(s):  
Sarah Page ◽  
Michael Salem ◽  
Maren R. Laughlin
Keyword(s):  
Adenine Nucleotide ◽  
Phosphoglycerate Kinase ◽  
Pentose Phosphate ◽  
Human Erythrocytes ◽  
Maximal Velocity ◽  
Nucleotide Synthesis ◽  
Ion Concentrations ◽  
Net Flux ◽  
Normal Fluctuations ◽  
Phosphorylation Rate

13C- and31P-NMR were used in methylene blue-treated human erythrocytes to determine the dependence on intracellular Mg2+concentration ([Mg2+]i) of the pentose phosphate pathway (PPP), the glycolytic pathway, and adenine nucleotide synthesis. The PPP flux had an [Mg2+]iat half-maximal velocity ([Mg2+]i,0.5) of 0.02 mM, well below the physiological range (0.2–0.7 mM). Flux through the PPP was reduced at higher [Mg2+]ias flux through phosphofructokinase was increased ([Mg2+]i,0.5= 0.16 mM). [Mg2+]i,0.5of phosphoglycerate kinase flux, which equals net ADP phosphorylation rate, was 0.27 mM, well within the physiological [Mg2+]irange. The rate of adenine nucleotide synthesis from [2-13C]glucose-derived ribose 5-phosphate and exogenous adenine also exhibited dependence on [Mg2+]ibut was not saturable up to 1.6 mM. Therefore, net flux through the PPP and glycolytic pathways in erythrocytes is not strongly dependent on [Mg2+]iat physiological ion concentrations, but both ADP phosphorylation and adenine nucleotide synthesis are likely to be regulated by normal fluctuations in [Mg2+]i.

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