Glucose consumption of vascular cell types in culture; toward optimization of experimental conditions

2021 ◽  
Author(s):  
Keiichi Torimoto ◽  
Keisuke Okuno ◽  
Ryohei Kuroda ◽  
No'Ad Shanas ◽  
Stephanie M. Cicalese ◽  
...  
Keyword(s):  
High Glucose ◽  
Glucose Concentration ◽  
Regulatory Protein ◽  
Glucose Consumption ◽  
Cell Types ◽  
Hexokinase Ii ◽  
Experimental Conditions ◽  
Vascular Cell ◽  
Adventitial Fibroblasts ◽  
Low Glucose

In this study, we have looked for an optimum media glucose concentration and compared glucose consumption in three vascular cell types, endothelial cells (EC), vascular smooth muscle cells (VSMC) and adventitial fibroblasts (AF) with or without angiotensin II (AngII) stimulation. In a sub-confluent 6-well experiment in 1 mL DMEM with a standard low (100 mg/dL), a standard high (450 mg/dL), or a mixed middle (275 mg/dL) glucose concentration, steady and significant glucose consumption was observed in all cell types. After 48-hour incubation, media that contained low glucose was reduced to almost 0 mg/dL, media that contained high glucose remained significantly higher at ~275 mg/dL, and media that contained middle glucose remained closer to physiological range. AngII treatment enhanced glucose consumption in AF and VSMC but not in EC. Enhanced extracellular acidification rate by AngII was also observed in AF. In AF, AngII induction of target proteins at 48 hours varied depending on the glucose concentration used. In low glucose media induction of glucose regulatory protein 78 or hexokinase II was highest, whereas induction of VCAM-1 was lowest. Utilization of specific inhibitors further suggest essential roles of AT1 receptor and glycolysis in AngII-induced fibroblast activation. Overall, the present study demonstrates a high risk of hypo- or hyperglycemic conditions when standard low or high glucose media is used with vascular cells. Moreover, these conditions may significantly alter experimental outcomes. Media glucose concentration should be monitored during any culture experiments and utilization of middle glucose media is recommended for all vascular cell types.

scholarly journals Effect of Substrate Feeding Concentration on Initial Biofilm Development in Anaerobic Hybrid Reactor

2017 ◽  
Vol 20 (3&4) ◽  
pp. 361-372
Author(s):  
B. Suraraksa ◽  
A. Nopharatana ◽  
P. Chaiprasert ◽  
S. Bhumiratana ◽  
M. Tanticharoen
Keyword(s):  
High Glucose ◽  
Glucose Concentration ◽  
Substrate Concentration ◽  
Early Stage ◽  
Hybrid Reactor ◽  
High Glucose Concentration ◽  
High Substrate Concentration ◽  
High Substrate ◽  
Low Glucose ◽  
Biofilm Development

To elucidate the effect of substrate concentration on biofilm development, glucose concentrations of 500 and 1,000 mg/L were used.  At an early stage, biofilm development at both concentrations was not significantly different (P=0.621).  After removing suspended biomass at 24 operational hours, the biofilm development at high substrate concentration was higher than at lower concentration.  At 72 operational hours, the amounts of attached biomass at low and high glucose feeding were 9.04±1.17 and 28.58±2.72 g VSS/m2, respectively.  The activities of acidogens, acetogens, and methanogens at the low glucose concentration were 0.334, 0.016 and 0.003 g COD/g VSS/h, and those at the high glucose concentration were 0.145, 0.003 and 0.001 g COD/g VSS/h, respectively.  Moreover, the ratio of methanogenic activity at low glucose concentration was higher than at high glucose concentration.  The glucose utilization at low and high feeding concentrations was 33% and 27%, respectively.  These results indicated that rapid biofilm development by using high substrate concentration would be less beneficial if unbalance of methanogenic ratio was found in biofilm.

Glucose stimulates O2 consumption, NOS, and Na/H exchange in diabetic rat proximal tubules

2002 ◽  
Vol 283 (2) ◽  
pp. F286-F293 ◽  
Author(s):  
Andrew Baines ◽  
Patrick Ho
Keyword(s):  
High Glucose ◽  
Glucose Concentration ◽  
Endothelial Nitric Oxide Synthase ◽  
Diabetic Rats ◽  
Proximal Tubules ◽  
Diabetic Rat ◽  
Uncontrolled Diabetes ◽  
Low Glucose ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide

Endothelial nitric oxide synthase (NOS) and neuronal NOS protein increased in proximal tubules of acidotic diabetic rats 3–5 wk after streptozotocin injection. NOS activity (citrulline production) was similar in nondiabetic and diabetic tubules incubated with low glucose (5 mM glucose + 20 mM mannitol); but after 30 min with high glucose (25 mM), Ca-sensitive citrulline production had increased 23% in diabetic tubules. Glucose concentration did not influence citrulline production in nondiabetic tubules. High glucose increased carboxy-2-phenyl-4,4,5,5,-tetramethylimidazoline 1-oxyl-3-oxide (cpt10)-scavenged NO sevenfold in a suspension of diabetic tubules but did not alter NO in nondiabetic tubules. Diabetes increased ouabain-sensitive 86Rb uptake (141 ± 9 vs. 122 ± 6 nmol · min−1 · mg−1) and oligomycin-sensitive O2 consumption (Q˙o 2; 16.0 ± 1.7 vs. 11.3 ± 0.7 nmol · min−1 · mg−1). Ethylisopropyl amiloride-inhibitable Q˙o 2(6.5 ± 0.6 vs. 2.4 ± 0.3 nmol · min−1 · mg−1) accounted for increased oligomycin-sensitiveQ˙o 2 in diabetic tubules. N G-monomethyl-l-arginine methyl ester (l-NAME) inhibited most of the increase in86Rb uptake and Q˙o 2 in diabetic tubules. l-NAME had little effect on nondiabetic tubules. Inhibition of Q˙o 2 by ethylisopropyl amiloride and l-NAME was only 5–8% additive. Uncontrolled diabetes for 3–5 wk increases NOS protein in proximal tubules and makes NOS activity sensitive to glucose concentration. Under these conditions, NO stimulates Na-K-ATPase andQ˙o 2 in proximal tubules.

scholarly journals Nitric Oxide Inhibits the Degradation of IRP2

2005 ◽  
Vol 25 (4) ◽  
pp. 1347-1353 ◽  
Author(s):  
Jian Wang ◽  
Guohua Chen ◽  
Kostas Pantopoulos
Keyword(s):  
Nitric Oxide ◽  
Iron Deficiency ◽  
Regulatory Protein ◽  
Cell Types ◽  
Iron Chelator ◽  
Experimental Conditions ◽  
Labile Iron Pool ◽  
Experimental Systems ◽  
Rich Domain ◽  
Iron Pool

ABSTRACT Iron-regulatory protein 2 (IRP2), a posttranscriptional regulator of iron metabolism, undergoes proteasomal degradation in iron-replete cells, while it is stabilized in iron deficiency or hypoxia. IRP2 also responds to nitric oxide (NO), as shown in various cell types exposed to pharmacological NO donors and in gamma interferon/lipopolysaccharide-stimulated macrophages. However, the diverse experimental systems have yielded conflicting results on whether NO activates or inhibits IRP2. We show here that a treatment of mouse B6 fibroblasts or human H1299 lung cancer cells with the NO-releasing drug S-nitroso-N-acetyl-penicillamine (SNAP) activates IRP2 expression. Moreover, the exposure of H1299 cells to SNAP leads to stabilization of hemagglutinin (HA)-tagged IRP2, with kinetics analogous to those elicited by the iron chelator desferrioxamine. Similar results were obtained with IRP2Δ 73, a mutant lacking a conserved, IRP2-specific proline- and cysteine-rich domain. Importantly, SNAP fails to stabilize HA-tagged p53, suggesting that under the above experimental conditions, NO does not impair the capacity of the proteasome for protein degradation. Finally, by employing a coculture system of B6 and H1299 cells expressing NO synthase II or IRP2-HA cDNAs, respectively, we demonstrate that NO generated in B6 cells stabilizes IRP2-HA in target H1299 cells by passive diffusion. Thus, biologically synthesized NO promotes IRP2 stabilization without compromising the overall proteasomal activity. These results are consistent with the idea that NO may negatively affect the labile iron pool and thereby trigger responses to iron deficiency.

scholarly journals A limited upstream region of the human sucrase-isomaltase gene confers glucose-regulated expression on a heterologous gene

1996 ◽  
Vol 315 (1) ◽  
pp. 301-306 ◽  
Author(s):  
Annie RODOLOSSE ◽  
Isabelle CHANTRET ◽  
Michel LACASA ◽  
Guillemette CHEVALIER ◽  
Alain ZWEIBAUM ◽  
...  
Keyword(s):  
Reporter Gene ◽  
High Glucose ◽  
Glucose Repression ◽  
Glucose Consumption ◽  
Human Colon ◽  
Upstream Region ◽  
Human Colon Carcinoma Cell ◽  
Repressive Effect ◽  
Hygromycin Resistance Gene ◽  
Low Glucose

We have previously shown that glucose can exert a repressive effect on the transcription of the sucrase–isomaltase (SI) gene in the differentiated enterocyte-like human colon carcinoma cell lines HT-29 and Caco-2. To characterize the region through which glucose exerts this effect, three different-length fragments of the 5´-flanking region of the human SI gene were linked to the reporter gene luciferase in an episomal vector carrying a hygromycin resistance gene. These fragments were used for transfection into a clone of the Caco-2 cell line, PF11, which has high glucose consumption and only expresses SI at high levels when cultured in the presence of a low supply of glucose. By using the stably transformed PF11 cells grown either in standard high glucose (25 mM) or in low glucose (1 mM) it was possible to show that the smallest fragment of the SI promoter, extending from bases -370 to +30, contains all the information required for the glucose repression of the reporter gene luciferase.

scholarly journals Glucose concentration during equine in vitro maturation alters mitochondrial function

Reproduction ◽  
2020 ◽  
Vol 160 (2) ◽  
pp. 227-237
Author(s):  
Niamh Lewis ◽  
Katrin Hinrichs ◽  
Henry J Leese ◽  
Caroline McGregor Argo ◽  
Daniel R Brison ◽  
...  
Keyword(s):  
Mitochondrial Function ◽  
High Glucose ◽  
Glucose Concentration ◽  
Mammalian Species ◽  
Glucose Consumption ◽  
Cumulus Cells ◽  
Metabolic Enzyme ◽  
Early Embryos ◽  
The Impact

The use of in vitro embryo production in the horse is increasing in clinical and research settings; however, protocols are yet to be optimised. Notably, the two most commonly used base media for in vitro maturation (IVM) supply glucose at markedly different concentrations: physiological (5.6 mM, M199) or supraphysiological (17 mM, DMEM/F-12). Exposure to high glucose has detrimental effects on oocytes and early embryos in many mammalian species, but the impact has not yet been examined in the horse. To address this, we compared the energy metabolism of equine COCs matured in M199-based maturation medium containing either 5.6 or 17 mM glucose, as well as expression of key genes in oocytes and cumulus cells. Oocytes were fertilised by ICSI and cultured. Analysis of spent medium revealed that COC glucose consumption and production of lactate and pyruvate were similar between treatments. However, the glycolytic index was decreased at 17 mM and analysis of mitochondrial function of COCs revealed that IVM in 17 mM glucose was associated with decreased ATP-coupled respiration and increased non-mitochondrial respiration compared to that for 5.6 mM glucose. We also found that the metabolic enzyme lactate dehydrogenase-A (LDHA) was downregulated in cumulus cells of oocytes that completed IVM in 17 mM glucose. There was no difference in maturation or blastocyst rates. These data indicate that COC mitochondrial function and gene expression are altered by high glucose concentration during IVM. Further work is needed to determine if these changes are associated with developmental changes in the resulting offspring.

scholarly journals Presence and differential expression of SGLT1, GLUT1, GLUT2, GLUT3 and GLUT5 hexose-transporter mRNAs in Caco-2 cell clones in relation to cell growth and glucose consumption

1994 ◽  
Vol 298 (3) ◽  
pp. 629-633 ◽  
Author(s):  
L Mahraoui ◽  
A Rodolosse ◽  
A Barbat ◽  
E Dussaulx ◽  
A Zweibaum ◽  
...  
Keyword(s):  
Cell Growth ◽  
Stationary Phase ◽  
High Glucose ◽  
Similar Pattern ◽  
Glucose Consumption ◽  
Glucose Deprivation ◽  
Close Correlation ◽  
Hexose Transporter ◽  
Low Glucose

Seven clones from the Caco-2 cell line, three isolated from passage 29 (PD7, PD10, PF11) and four from passage 198 (TB10, TC7, TF3, TG6), all of them selected on the basis of differences in the levels of expression of sucrase-isomaltase and rates of glucose consumption, were analysed for the expression of hexose-transporter mRNAs (SGLT1, GLUT1-GLUT5) in relation to the phases of cell growth and the associated variations of the rates of glucose consumption. All clones showed a similar pattern of evolution of the rates of glucose consumption, which decreased from the exponential to the late-stationary phase, but differed, in a 1-40-fold range, in the values observed at late postconfluency. According to these values, clones could be divided into high- (PD10, PF11) and low-glucose-consuming cells (PD7, TB10, TC7, TF3 and TG6). GLUT1 and GLUT3 mRNAs were expressed in all clones and showed a similar pattern of evolution: their level decreased, from the exponential to the stationary phase, in close correlation with the decrease in rates of glucose consumption, with only high-glucose-consuming clones maintaining high levels in the stationary phase. In contrast, SGLT1, GLUT2 and GLUT5 mRNAs were only expressed, like sucrase-isomaltase mRNA, in the low-glucose-consuming clones, and their level increased from the exponential to the stationary phase, in parallel with the differentiation of the cells. GLUT4 was undetectable in all the clones. Glucose deprivation generally resulted in a discrete decrease in the levels of all transporter mRNAs in all clones, one exception being GLUT2, which in the high-glucose-consuming clones is only detectable when the cells are grown in low glucose. These clones should be ideal tools with which to study in vitro, at the single-cell level, how these transporters concur to the utilization and transport of hexoses and how their exclusive or co-ordinated expression is regulated.

scholarly journals Evaluation of Cells and Medium Optimization for Invitro Model of Diabetic and Electrolyte Imbalance

2021 ◽  
Vol 41 ◽  
pp. 05003
Author(s):  
Alfino Sebastian ◽  
Widya Wasityastuti ◽  
Dwi Aris Nugrahaningsih ◽  
Hevi Wihadmadyatami ◽  
Tutik Sri Wahyuni ◽  
...  
Keyword(s):  
High Glucose ◽  
Glucose Concentration ◽  
Gene Knockout ◽  
Living Cells ◽  
Electrolyte Imbalance ◽  
Live Cells ◽  
Glucose Medium ◽  
Knock Out ◽  
Low Glucose ◽  
The Right

Metabolism syndrome has many negative impacts on human health. Various efforts and methods are attempted in the treatment of this disease. One of the methods used is CRISPR/Cas9 gene therapy. Re-testing of knock out cells using the CRISPR/Cas9 method is needed to evaluate its success. In conducting the test, the right medium is needed so that the results are optimal and can be evaluated properly. In this study, we optimized the medium for three types of cells (fibroblasts, myoblasts and macrophages) in high and low glucose medium to evaluate gene knockout results. The medium was modified by adding high concentrations of glucose and sodium. The results, in macrophage culture, giving variations in glucose concentration in low glucose medium gave a significantly different percentage of live cells between treatments, while the treatment with variations in glucose concentration in macrophages in high glucose medium and fibroblasts and myoblasts in high and low glucose medium did not show any difference in the percentage of living cells. In the treatment of various concentrations of natrium, macrophages, fibroblasts and myoblasts on high and low glucose medium all showed significantly different percentages of living cells. Therefore, DMEM low glucose medium is suitable as a medium for the treatment of high glucose and natrium induction in macrophage cells, but is not suitable for fibroblast and myoblast cells.

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.

Online Measurement of Glucose Consumption from HepG2 Cells Using an Integrated Bioreactor and Enzymatic Assay

2018 ◽  
Author(s):  
Anna Adams ◽  
Radha Krishna Murthy Bulusu ◽  
Nikita Mukhitov ◽  
Jose Mendoza-Cortes ◽  
Michael Roper
Keyword(s):  
Real Time ◽  
Glucose Concentration ◽  
Hepg2 Cells ◽  
Glucose Consumption ◽  
Microfluidic System ◽  
Structure And Function ◽  
Online Measurement ◽  
Fluorescent Assay ◽  
And Function ◽  
Integrated Bioreactor

In this work, we developed a microfluidic bioreactor for optimizing growth and maintaining structure and function of HepG2, and when desired, the device could be removed and the extracellular output from the bioreactor combined with enzymatic glucose reagents into a droplet-based microfluidic system. The intensity of the resulting fluorescent assay product in the droplets was measured, and was directly correlated to glucose concentration, allowing the effect of insulin on glucose consumption in the HepG2 cells to be observed and quantified online and in near real-time.

Online Measurement of Glucose Consumption from HepG2 Cells Using an Integrated Bioreactor and Enzymatic Assay

2018 ◽  
Author(s):  
Anna Adams ◽  
Radha Krishna Murthy Bulusu ◽  
Nikita Mukhitov ◽  
Jose Mendoza-Cortes ◽  
Michael Roper
Keyword(s):  
Real Time ◽  
Glucose Concentration ◽  
Hepg2 Cells ◽  
Glucose Consumption ◽  
Microfluidic System ◽  
Structure And Function ◽  
Online Measurement ◽  
Fluorescent Assay ◽  
And Function ◽  
Integrated Bioreactor

In this work, we developed a microfluidic bioreactor for optimizing growth and maintaining structure and function of HepG2, and when desired, the device could be removed and the extracellular output from the bioreactor combined with enzymatic glucose reagents into a droplet-based microfluidic system. The intensity of the resulting fluorescent assay product in the droplets was measured, and was directly correlated to glucose concentration, allowing the effect of insulin on glucose consumption in the HepG2 cells to be observed and quantified online and in near real-time.

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