Enzyme activity levels underestimate lactate production rates in cod (Gadus morhua) gas gland

1990 ◽  
Vol 68 (1) ◽  
pp. 193-197 ◽  
Author(s):  
H. Stephen Ewart ◽  
William R. Driedzic
Keyword(s):  
Metabolic Profile ◽  
Gadus Morhua ◽  
Citrate Synthase ◽  
Lactate Production ◽  
Low Ph ◽  
Activity Levels ◽  
Aerobic Incubation ◽  
Hydroxyacyl Coa Dehydrogenase ◽  
Gas Gland

Maximal in vitro activities of hexokinase, phosphofructokinase, pyruvate kinase, lactate dehydrogenase, β-hydroxyacyl-CoA dehydrogenase, citrate synthase, malate dehydrogenase, and cytochrome oxidase were assessed in cod (Gadus morhua) gas gland. The metabolic profile predicts a substantial anaerobic relative to aerobic metabolism. The effect of catecholamines, acetylcholine, and low pH on in vitro rates of lactate production by gas gland was assessed. Adrenaline and acetylcholine both increased the rate of lactate production even under aerobic incubation conditions. However, the rates of lactate production were well below the capacity suggested by the enzyme levels. It is suggested that the tissue has an abundance of enzymes that operate at submaximal rates.

THE EFFECTS OF LOW PH ON CORNEAL LACTATE PRODUCTION IN VITRO

1995 ◽  
Vol 72 (SUPPLEMENT) ◽  
pp. 229
Author(s):  
Susan Lin ◽  
Arlene Sarroca ◽  
Joseph A. Bonanno
Keyword(s):  
Lactate Production ◽  
Low Ph

Metabolic Acidosis is a Potent Stimulus for Cellular Inorganic Phosphate Generation in Uraemia

1995 ◽  
Vol 88 (4) ◽  
pp. 405-412 ◽  
Author(s):  
Alan Bevington ◽  
Dennis Brough ◽  
Frease E. Baker ◽  
Jane Hattersley ◽  
John Walls
Keyword(s):  
Steady State ◽  
Metabolic Acidosis ◽  
Inorganic Phosphate ◽  
Lactate Production ◽  
Extracellular Fluid ◽  
Low Ph ◽  
Organic Phosphate ◽  
Glycolytic Flux ◽  
Before And After

1. During metabolic acidosis, significant fluxes of inorganic phosphate (Pi) may occur from cellular to extracellular fluid. In this study Pi was measured in erythrocytes of uraemic patients before and after haemodialysis and was related to their plasma pH (acidosis), plasma Pi (hyperphosphataemia) and cellular organic phosphate concentrations. 2. Before dialysis, the ratio of cellular to extracellular Pi concentration correlated inversely with plasma pH, increasing 2.5-fold as pH fell from 7.4 to 7.2. 3. An increase in cellular Pi similar to that seen in the patients was observed within 90 min of adding acid to normal erythrocytes in vitro. 4. The total Pi content of the cell suspension increased 25% on decreasing plasma pH from 7.4 to 7.2, largely as a result of generation of Pi from 2,3-bisphosphoglycerate in the cells. This was accompanied by net efflux of Pi into plasma. 5. In addition, the increase in the steady-state cellular Pi concentration on adding a constant extracellular Pi load was 50% greater at pH 7.2 than at 7.4, implying that alterations in the regulation of the transmembrane Pi gradient also contribute to the rise in cellular Pi observed at low pH. 6. At normal plasma Pi concentration (1 mM), glycolytic flux (lactate production) was inhibited by 20% when pH was lowered from 7.4 to 7.2. However, this inhibition was blocked when cellular Pi was increased by adding Pi to the plasma in vitro. 7. Metabolic acidosis is therefore a potent stimulus for Pi generation in erythrocytes, and this Pi may serve to stimulate glycolysis which is normally inhibited by low pH.

The Effects of a Theacrine-based Supplement on mRNAs Related to Various Metabolic Processes and Sirtuin Activity in vitro

2020 ◽  
Author(s):  
Petey Mumford ◽  
Shelby Osburn ◽  
Michael D. Roberts
Keyword(s):  
Mitochondrial Biogenesis ◽  
Citrate Synthase ◽  
Inflammatory Marker ◽  
C2c12 Myoblast ◽  
Activity Levels ◽  
Mrna Levels ◽  
Expression Of Genes ◽  
Myoblast Cell Line ◽  
Myoblast Cell

Abstract There is evidence in rodents to suggest theacrine-based supplements modulate tissue sirtuin activity as well as other biological processes associated with aging. Herein, we examined if a theacrine-based supplement (NAD3) altered sirtuin activity in vitro while also affecting markers of mitochondrial biogenesis and the mRNA expression of genes related to various cellular processes in muscle. The murine C2C12 myoblast cell line was used for experimentation. Following 7 days of differentiation, myotubes were treated with 0.45 mg/mL of NAD3 (containing ~ 2 mM theacrine) for 3 and 24 hours (n=6 treatment wells per time point). Control treatments consisted of cellulose-only treatments at the same time points. Relative to CTL-treated cells, NAD3 treatments increased (p<0.05) Sirt1 mRNA levels at 3 hours, as well as global sirtuin activity at 3 and 24 hours. While NAD3 treatments decreased mRNA levels of Nfe2l2 at 3 hours and increased levels at 24 hours relative to CTL-treated cells (a gene involved in mitochondrial biogenesis, p<0.05), citrate synthase activity levels (a surrogate of mitochondrial density) remained unaltered between treatments. NAD3 treatments for 3 and 24 hours decreased Nlrp3 mRNA levels relative to CTL-treated cells (an inflammatory marker, p<0.05). Additionally, NAD3 treatments decreased Map1lc3b mRNA levels (an autophagy marker) after 24-hour treatments (p<0.05). Although these data are limited to select biomarkers in vitro, these preliminary findings suggest a theacrine-based supplement can modulate various skeletal muscle biomarkers related to sirtuin activity, inflammation, and autophagy. Muscle biopsy studies in humans are needed to confirm these current findings.

Human skeletal muscle adaptation in response to chronic low-frequency electrical stimulation

1994 ◽  
Vol 77 (4) ◽  
pp. 1885-1889 ◽  
Author(s):  
R. Theriault ◽  
G. Theriault ◽  
J. A. Simoneau
Keyword(s):  
Skeletal Muscle ◽  
Electrical Stimulation ◽  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Human Skeletal Muscle ◽  
Citrate Synthase ◽  
Low Frequency ◽  
Vastus Lateralis Muscle ◽  
Activity Levels ◽  
Hydroxyacyl Coa Dehydrogenase

The purpose of the study was to verify the influence of several weeks of chronic low-frequency electrical stimulation (LFES) on the metabolic profile and functional capacity of human skeletal muscle. Knee extensor muscles (KEM) of eight subjects were electrically stimulated at 8 Hz for 8 h/day and 6 days/wk. Vastus lateralis muscle samples were taken before, after 4 wk, and after 8 wk of LFES, and activities of anaerobic (creatine kinase, phosphofructokinase, glyceraldehyde-3-phosphate dehydrogenase) and aerobic-oxidative (citrate synthase, 3-hydroxyacyl-CoA dehydrogenase, cytochrome-c oxidase) enzyme markers were determined. KEM dynamic performance was also assessed before, after 4 wk, and after 8 wk of LFES. Activity levels of anaerobic enzymes were not altered, whereas the activity levels of citrate synthase (29%),3-hydroxyacyl-CoA dehydrogenase (22%), and cytochrome-c oxidase (25%) were significantly increased after 4 wk of LFES but were not further increased after 4 additional wk of LFES. KEM performance was also improved (P < 0.05) but leveled off after 4 wk of LFES. Although significant changes were observed, the results of the present study suggest that the muscle characteristics investigated in the current study have a limited capacity of adaptation in response to this form of chronic LFES.

scholarly journals Enzymic and metabolic adaptations in the gastrocnemius, plantaris and soleus muscles of hypocaloric rats

1989 ◽  
Vol 261 (1) ◽  
pp. 219-225 ◽  
Author(s):  
M S M Ardawi ◽  
M F Majzoub ◽  
I M Masoud ◽  
E A Newsholme
Keyword(s):  
Citrate Synthase ◽  
Muscle Relaxation ◽  
Lactate Production ◽  
Relaxation Rates ◽  
High Frequencies ◽  
Metabolic Adaptations ◽  
Hydroxyacyl Coa Dehydrogenase ◽  
Oxoglutarate Dehydrogenase ◽  
Frequency Of Stimulation ◽  
Normal Food

1. The effect of hypocaloric feeding (25% of normal food intake for 21 days) of rats on the enzymic and metabolic adaptations in the gastrocnemius, plantaris and soleus muscles was studied. 2. In control and hypocaloric rats the muscle relaxation rates at 100 Hz were 35.76 and 11.38% force loss/10 ms respectively. Control rats exhibited enhanced force of muscle contraction as the frequency of stimulation increased from 10 to 100 Hz, with maximum force being at 100 Hz. Hypocaloric rats exhibited a decrease in the increment of force being exerted at high frequencies, with maintenance of force at lower stimulatory frequencies. 3. In muscles of hypocaloric rats, there were significant decreases in the maximal activities of hexokinase (17.6-37.0%), 6-phosphofructokinase (22.7-34.2%), pyruvate kinase (21.2-36.0%), citrate synthase (34.1-41.5%), oxoglutarate dehydrogenase (29.4-52.4%) and 3-hydroxyacyl-CoA dehydrogenase (26.7-32.1%), whereas the activities of glycogen phosphorylase increased (23.8-43.4%) compared with control values. 4. In soleus-muscle strip preparations of hypocaloric rats, there were significant decreases in the rates of lactate production (28.1%) and glucose oxidation (32.6%) compared with control preparations. 5. Mitochondrial preparations from muscles of hypocaloric rats incubated with various substrates exhibited decreased rates of oxygen uptake compared with control preparations. 6. In muscles of hypocaloric rats (gastrocnemius and soleus), there were significant decreases in the concentrations of glycogen (P less than 0.001) and phosphocreatine (P less than 0.001) and increases in those of pyruvate (P less than 0.001), lactate (P less than 0.001) and ADP (P less than 0.001), whereas those of ATP and AMP remained unchanged. 7. Calculated [lactate]/[pyruvate] and [ATP]/[ADP] ratios exhibited significant increases (P less than 0.05) and decreases (P less than 0.05) in muscles of hypocaloric rats respectively. 8. The results are discussed in relation to the genesis of muscle dysfunction caused by malnutrition.

scholarly journals Regulation of the Elevated T Cell Glycolysis May Alleviate Acute Graft-Versus-Host Disease Post-Allotransplant

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 600-600
Author(s):  
Qi Wen ◽  
Song Yang ◽  
Zhong-Shi Lyu ◽  
Wei-Li Yao ◽  
Yu-Hong Chen ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Metabolic Profile ◽  
Lactate Production ◽  
Glucose Consumption ◽  
Metabolic State ◽  
Glycolytic Activity ◽  
Abnormal Metabolism ◽  
Glycolysis Inhibitor

Background: Acute graft-versus-host disease(aGVHD) remains a major complication following allogeneic hematopoietic stem cell transplantation(allo-HSCT). The pathogenesis of aGVHD is commonly considered to be caused by exaggerated and undesirable immune responses. Metabolism not only provide energy and substrates for T cell growth and survival, but also instruct effector functions, differentiation, and gene expression of T cells. In this regard, the metabolic profile of T cells was reported to play a critical role in the occurrence and development of many immunological disorders such as systemic lupus erythematosus and rheumatoid arthritis. Murine studies found that alloreactive T cells use aerobic glycolysis as the predominant metabolic process to meet activation and proliferation demand after allo-HSCT. However, the metabolic profile of T cells and the approach for regulating T cell metabolism in aGVHD patients remains to be elucidated. Aims: To determine the metabolic state in T cells of patients with aGVHD. Moreover, to investigate the effect of the novel approach targeting the abnormal metabolism in T cells of aGVHD patients, which may provide a potential therapeutic target for aGVHD patients after allo-HSCT. Methods: In this prospective case-control study, a total of 25 patients with aGVHD and 25 matched patients without aGVHD(non-aGVHD) after allo-HSCT were enrolled. T cell subsets were analyzed in aGVHD and non-aGVHD patients by flow cytometry. Th1, Th2, Th17, and Treg cells were identified as CD4+IFN-γ+, CD4+IL-4+, CD4+IL17A+, and CD4+CD25+Foxp3+, respectively. Tc1 and Tc2 cells were identified as CD8+IFN-γ+ and CD8+IL-4+, respectively. In order to determine the metabolic state in T cells of patients with aGVHD and non-aGVHD, the metabolic profile was determined using a Seahorse XF96 Analyzer. The glucose consumption and lactate production rates were detected by glucose assay kit and lactate assay kit. The mitochondrial mass, the mitochondrial membrane potential, the protein expressions for the lipid metabolism enzyme CTP1a and the glycolytic activator PFKFB3 were measured by flow cytometry. To further understand the metabolic state of T cells in aGVHD and non-aGVHD patients and investigate its mechanism, RNA sequencing (RNA-Seq) was performed to analyze the gene expression profiles of T cells. Subsequently, to explore the potential way of targeting the abnormal metabolism in T cells, the glycolysis inhibitor 3-PO was administrated to T cells from aGVHD patients. Results: When compared with T cells in non-aGVHD patients, T cells in aGVHD patients were polarized towards pro-inflammatory T cells, characterized by an elevated proportion of Tc1, Th1 and Th17. Furthermore, T cells isolated from aGVHD patients exhibited higher extracellular acidification rate, as well as the increased glucose consumption rate and lactate production rate compared to those in non-aGVHD patients. Moreover, elevated expression of PFKFB3 was observed in T cells, especially in naïve T cells of aGVHD patients, but oxygen consumption rate, CPT1A, mitochondrial mass or membrane potential showed no significant differences in T cells between aGVHD and non-aGVHD patients. These results implied higher glycolytic activity of T cells in aGVHD patients when compared with those in non-aGVHD patients. Consistent with the increased glycolytic activity observed in T cells from aGVHD patients, the mRNA levels of genes involved in the glycolytic pathway were substantially elevated in T cells of aGVHD patients compared to those in non-aGVHD patients. Importantly, in vitro treatment with glycolysis inhibitor 3-PO improved the activity of T cells derived from aGVHD patients through down-regulating glycolytic activity of T cells. Summary/Conclusion: The current study demonstrated that T cells in aGVHD patients preferentially depend on glycolysis to meet activation and proliferation demands. Furthermore, the activity of T cells from aGVHD patients could be ameliorated by glycolysis inhibitor 3-PO in vitro. Although further validation is required, T cell glycolysis promises to be a novel therapeutic target for aGVHD patients after allo-HSCT. Disclosures No relevant conflicts of interest to declare.

ÉTUDE IN VITRO DU MÉTABOLISME DES HYDRATES DE CARBONE DANS LE LEUCOCYTE CIRCULANT DU COBAYE TRAITÉ À LA CORTISONE

1966 ◽  
Vol 51 (2) ◽  
pp. 193-202
Author(s):  
J. A. Antonioli ◽  
A. Vannotti
Keyword(s):  
Glucose Concentration ◽  
Intramuscular Injection ◽  
Acute Inflammation ◽  
Glycogen Content ◽  
Glycogen Synthesis ◽  
Lactate Production ◽  
Glucose Consumption ◽  
List Type ◽  
Hydrates De Carbone

ABSTRACT 1. The metabolism of suspensions of circulating leucocytes has been studied after intramuscular injection of a dose of 50 mg/kg of a corticosteroid (cortisone acetate). The suspensions were incubated under aerobic conditions in the presence of a glucose concentration of 5.6 mm. Glucose consumption, lactate production, and variations in intracellular glycogen concentration were measured. After the administration of the corticosteroid, the anabolic processes of granulocyte metabolism were reversibly stimulated. Glucose consumption and lactate production increased 12 hours after the injection, but tended to normalize after 24 hours. The glycogen content of the granulocytes was enhanced, and glycogen synthesis during the course of the incubation was greatly stimulated. The action of the administered corticosteroid is more prolonged in females than in males. The injection of the corticosteroid caused metabolic modifications which resemble in their modulations and in their chronological development those found in circulating granulocytes of guinea-pigs suffering from sterile peritonitis. These results suggest, therefore, that, in the case of acute inflammation, the glucocorticosteroids may play an important role in the regulation of the metabolism of the blood leucocytes.

scholarly journals Preservation of Human Gut Microbiota Inoculums for In Vitro Fermentations Studies

Fermentation ◽  
2021 ◽  
Vol 7 (1) ◽  
pp. 14
Author(s):  
Nelson Mota de Carvalho ◽  
Diana Luazi Oliveira ◽  
Mayra Anton Dib Saleh ◽  
Manuela Pintado ◽  
Ana Raquel Madureira
Keyword(s):  
Gut Microbiota ◽  
Metabolic Profile ◽  
Bacterial Count ◽  
Glycerol Solution ◽  
Metabolic Profiles ◽  
Human Gut ◽  
Colonic Fermentation ◽  
In Vitro Fermentation ◽  
Fecal Samples

The use of fecal inoculums for in vitro fermentation models requires a viable gut microbiota, capable of fermenting the unabsorbed nutrients. Fresh samples from human donors are used; however, the availability of fresh fecal inoculum and its inherent variability is often a problem. This study aimed to optimize a method of preserving pooled human fecal samples for in vitro fermentation studies. Different conditions and times of storage at −20 °C were tested. In vitro fermentation experiments were carried out for both fresh and frozen inoculums, and the metabolic profile compared. In comparison with the fresh, the inoculum frozen in a PBS and 30% glycerol solution, had a significantly lower (p < 0.05) bacterial count (<1 log CFU/mL). However, no significant differences (p < 0.05) were found between the metabolic profiles after 48 h. Hence, a PBS and 30% glycerol solution can be used to maintain the gut microbiota viability during storage at −20 °C for at least 3 months, without interfering with the normal course of colonic fermentation.

scholarly journals Warburg effect-promoted exosomal circ_0072083 releasing up-regulates NANGO expression through multiple pathways and enhances temozolomide resistance in glioma

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Chenyu Ding ◽  
Xuehan Yi ◽  
Xiangrong Chen ◽  
Zanyi Wu ◽  
Honghai You ◽  
...  
Keyword(s):  
Warburg Effect ◽  
Lactate Production ◽  
Xenograft Model ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Circular Rnas ◽  
Rna Immunoprecipitation ◽  
Resistant Cells

Abstract Background Temozolomide (TMZ) resistance limits its application in glioma. Exosome can carry circular RNAs (circRNAs) to regulate drug resistance via sponging microRNAs (miRNAs). miRNAs can control mRNA expression by regulate the interaction with 3’UTR and methylation. Nanog homeobox (NANOG) is an important biomarker for TMZ resistance. Hitherto, it is unknown about the role of exosomal hsa_circ_0072083 (circ_0072083) in TMZ resistance in glioma, and whether it is associated with NANOG via regulating miRNA sponge and methylation. Methods TMZ-resistant (n = 36) and sensitive (n = 33) patients were recruited. The sensitive cells and constructed resistant cells were cultured and exposed to TMZ. circ_0072083, miR-1252-5p, AlkB homolog H5 (ALKBH5) and NANOG levels were examined via quantitative reverse transcription polymerase chain reaction and western blot. The half maximal inhibitory concentration (IC50) of TMZ, cell proliferation, apoptosis, migration and invasion were analyzed via Cell Counting Kit-8, colony formation, flow cytometry, wound healing and transwell assays. The in vivo function was assessed using xenograft model. The N6-methyladenosine (m6A) level was analyzed via methylated RNA immunoprecipitation (MeRIP). Target relationship was investigated via dual-luciferase reporter assay and RNA immunoprecipitation. Warburg effect was investigated via lactate production, glucose uptake and key enzymes expression. Exosome was isolated and confirmed via transmission electron microscopy and specific protein expression. Results circ_0072083 expression was increased in TMZ-resistant glioma tissues and cells. circ_0072083 knockdown restrained the resistance of resistant cells via decreasing IC50 of TMZ, proliferation, migration, invasion and xenograft tumor growth and increasing apoptosis. circ_0072083 silence reduced NANOG expression via blocking ALKBH5-mediated demethylation. circ_0072083 could regulate NANOG and ALKBH5 via targeting miR-1252-5p to control TMZ resistance. Warburg effect promoted the release of exosomal circ_0072083 in resistant cells. Exosomal circ_0072083 from resistant cells increased the resistance of sensitive cells to TMZ in vitro and xenograft model. Exosomal circ_0072083 level was enhanced in resistant patients, and it had a diagnostic value and indicated a lower overall survival in glioma. Conclusion Exosomal circ_0072083 promoted TMZ resistance via increasing NANOG via regulating miR-1252-5p-mediated degradation and demethylation in glioma.

scholarly journals Bone marrow mesenchymal stem cells promote prostate cancer cell stemness via cell–cell contact to activate the Jagged1/Notch1 pathway

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Ji-wen Cheng ◽  
Li-xia Duan ◽  
Yang Yu ◽  
Pu Wang ◽  
Jia-le Feng ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Western Blot ◽  
Cell Contact ◽  
Activity Levels ◽  
Tumor Resistance ◽  
Cell Cell

Abstract Background Mesenchymal stem cells (MSCs) play a crucial role in cancer development and tumor resistance to therapy in prostate cancer, but the influence of MSCs on the stemness potential of PCa cells by cell–cell contact remains unclear. In this study, we investigated the effect of direct contact of PCa cells with MSCs on the stemness of PCa and its mechanisms. Methods First, the flow cytometry, colony formation, and sphere formation were performed to determine the stemness of PCaMSCs, and the expression of stemness-related molecules (Sox2, Oct4, and Nanog) was investigated by western blot analysis. Then, we used western blot and qPCR to determine the activity levels of two candidate pathways and their downstream stemness-associated pathway. Finally, we verified the role of the significantly changed pathway by assessing the key factors in this pathway via in vitro and in vivo experiments. Results We established that MSCs promoted the stemness of PCa cells by cell–cell contact. We here established that the enhanced stemness of PCaMSCs was independent of the CCL5/CCR5 pathway. We also found that PCaMSCs up-regulated the expression of Notch signaling-related genes, and inhibition of Jagged1-Notch1 signaling in PCaMSCs cells significantly inhibited MSCs-induced stemness and tumorigenesis in vitro and in vivo. Conclusions Our results reveal a novel interaction between MSCs and PCa cells in promoting tumorigenesis through activation of the Jagged1/Notch1 pathway, providing a new therapeutic target for the treatment of PCa.

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