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.

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 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 Glucose-deprivation increases thyroid cancer cells sensitivity to metformin

2015 ◽  
Vol 22 (6) ◽  
pp. 919-932 ◽  
Author(s):  
Athanasios Bikas ◽  
Kirk Jensen ◽  
Aneeta Patel ◽  
John Costello ◽  
Dennis McDaniel ◽  
...  
Keyword(s):  
Cell Death ◽  
Thyroid Cancer ◽  
Cancer Cell ◽  
High Glucose ◽  
Glucose Medium ◽  
Thyroid Cancer Cell ◽  
High Glucose Medium ◽  
Anti Cancer ◽  
Low Glucose ◽  
Thyroid Cancer Cells

Metformin inhibits thyroid cancer cell growth. We sought to determine if variable glucose concentrations in medium alter the anti-cancer efficacy of metformin. Thyroid cancer cells (FTC133 and BCPAP) were cultured in high-glucose (20 mM) and low-glucose (5 mM) medium before treatment with metformin. Cell viability and apoptosis assays were performed. Expression of glycolytic genes was examined by real-time PCR, western blot, and immunostaining. Metformin inhibited cellular proliferation in high-glucose medium and induced cell death in low-glucose medium. In low-, but not in high-glucose medium, metformin induced endoplasmic reticulum stress, autophagy, and oncosis. At micromolar concentrations, metformin induced phosphorylation of AMP-activated protein kinase and blocked p-pS6 in low-glucose medium. Metformin increased the rate of glucose consumption from the medium and prompted medium acidification. Medium supplementation with glucose reversed metformin-inducible morphological changes. Treatment with an inhibitor of glycolysis (2-deoxy-d-glucose (2-DG)) increased thyroid cancer cell sensitivity to metformin. The combination of 2-DG with metformin led to cell death. Thyroid cancer cell lines were characterized by over-expression of glycolytic genes, and metformin decreased the protein level of pyruvate kinase muscle 2 (PKM2). PKM2 expression was detected in recurrent thyroid cancer tissue samples. In conclusion, we have demonstrated that the glucose concentration in the cellular milieu is a factor modulating metformin's anti-cancer activity. These data suggest that the combination of metformin with inhibitors of glycolysis could represent a new strategy for the treatment of thyroid cancer.

High ambient glucose is effect neutral on cell death and proliferation in human proximal tubular epithelial cells

2005 ◽  
Vol 289 (2) ◽  
pp. F401-F409 ◽  
Author(s):  
Christudas Morais ◽  
Justin Westhuyzen ◽  
Betty Pat ◽  
Glenda Gobe ◽  
Helen Healy
Keyword(s):  
Epithelial Cells ◽  
High Glucose ◽  
Culture Media ◽  
Primary Cultures ◽  
Cellular Growth ◽  
Glucose Medium ◽  
Tubular Epithelial Cells ◽  
Proximal Tubular Epithelial Cells ◽  
Proximal Tubular ◽  
Low Glucose

In vitro models of diabetic nephropathy that assess the role of hyperglycemia on proximal tubular cell turnover commonly compare cells in a high-glucose medium (25 or 30 mM) with a low-glucose medium (5 to 6.1 mM). Any cellular growth changes observed are usually attributed to the effect of high glucose. We hypothesize that in such experiments, glucose concentrations in the low-glucose medium may decline during the course of the experiments to levels that inhibit cell growth leading to the comparative conclusion that high glucose induces hyperplasia and/or hypertrophy. In this study, primary cultures of human proximal tubular epithelial cells (PTEC) and immortalized HK-2 cells were exposed to low (5 mM) or high (17, 30, or 47 mM) glucose for up to 6 days (PTEC) and 48 h (HK-2). When culture media were not replenished, low glucose induced a significant increase in necrosis and release of lactate dehydrogenase and a decrease in proliferation, metabolic activity, and protein content without any changes in apoptosis. High-glucose media failed to induce any of these changes. Glucose was undetectable in the low-glucose culture medium after 72 h. No significant differences were observed between any of the treatment groups when culture media were replenished daily. We conclude that regular replenishment of culture media is necessary to prevent the emergence of artifactual and misleading differences between high- and low-glucose groups. The current knowledge of the pathophysiology of high glucose based on cell culture systems may need to be reevaluated.

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 Excess Glucose Impedes the Proliferation of Skeletal Muscle Satellite Cells Under Adherent Culture Conditions

2021 ◽  
Vol 9 ◽  
Author(s):  
Yasuro Furuichi ◽  
Yuki Kawabata ◽  
Miho Aoki ◽  
Yoshitaka Mita ◽  
Nobuharu L. Fujii ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Stem Cells ◽  
Cell Fate ◽  
Satellite Cells ◽  
High Glucose ◽  
Glucose Concentration ◽  
Culture Conditions ◽  
Glucose Medium ◽  
High Glucose Medium ◽  
Adherent Culture

Glucose is a major energy source consumed by proliferating mammalian cells. Therefore, in general, proliferating cells have the preference of high glucose contents in extracellular environment. Here, we showed that high glucose concentrations impede the proliferation of satellite cells, which are muscle-specific stem cells, under adherent culture conditions. We found that the proliferation activity of satellite cells was higher in glucose-free DMEM growth medium (low-glucose medium with a glucose concentration of 2 mM) than in standard glucose DMEM (high-glucose medium with a glucose concentration of 19 mM). Satellite cells cultured in the high-glucose medium showed a decreased population of reserve cells, identified by staining for Pax7 expression, suggesting that glucose concentration affects cell fate determination. In conclusion, glucose is a factor that decides the cell fate of skeletal muscle-specific stem cells. Due to this unique feature of satellite cells, hyperglycemia may negatively affect the regenerative capability of skeletal muscle myofibers and thus facilitate sarcopenia.

scholarly journals Glucose regulates tissue-specific chondro-osteogenic differentiation of human cartilage endplate stem cells via O-GlcNAcylation of Sox9 and Runx2

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Chao Sun ◽  
Weiren Lan ◽  
Bin Li ◽  
Rui Zuo ◽  
Hui Xing ◽  
...  
Keyword(s):  
Western Blot ◽  
Osteogenic Differentiation ◽  
High Glucose ◽  
Immunohistochemical Staining ◽  
Chondrogenic Differentiation ◽  
Glucose Medium ◽  
Cartilage Endplate ◽  
High Glucose Medium ◽  
Differentiation Medium ◽  
Low Glucose

Abstract Background The degenerative disc disease (DDD) is a major cause of low back pain. The physiological low-glucose microenvironment of the cartilage endplate (CEP) is disrupted in DDD. Glucose influences protein O-GlcNAcylation via the hexosamine biosynthetic pathway (HBP), which is the key to stem cell fate. Thiamet-G is an inhibitor of O-GlcNAcase for accumulating O-GlcNAcylated proteins while 6-diazo-5-oxo-l-norleucine (DON) inhibits HBP. Mechanisms of DDD are incompletely understood but include CEP degeneration and calcification. We aimed to identify the molecular mechanisms of glucose in CEP calcification in DDD. Methods We assessed normal and degenerated CEP tissues from patients, and the effects of chondrogenesis and osteogenesis of the CEP were determined by western blot and immunohistochemical staining. Cartilage endplate stem cells (CESCs) were induced with low-, normal-, and high-glucose medium for 21 days, and chondrogenic and osteogenic differentiations were measured by Q-PCR, western blot, and immunohistochemical staining. CESCs were induced with low-glucose and high-glucose medium with or without Thiamet-G or DON for 21 days, and chondrogenic and osteogenic differentiations were measured by Q-PCR, western blot, and immunohistochemical staining. Sox9 and Runx2 O-GlcNAcylation were measured by immunofluorescence. The effects of O-GlcNAcylation on the downstream genes of Sox9 and Runx2 were determined by Q-PCR and western blot. Results Degenerated CEPs from DDD patients lost chondrogenesis, acquired osteogenesis, and had higher protein O-GlcNAcylation level compared to normal CEPs from LVF patients. CESC chondrogenic differentiation gradually decreased while osteogenic differentiation gradually increased from low- to high-glucose differentiation medium. Furthermore, Thiamet-G promoted CESC osteogenic differentiation and inhibited chondrogenic differentiation in low-glucose differentiation medium; however, DON acted opposite role in high-glucose differentiation medium. Interestingly, we found that Sox9 and Runx2 were O-GlcNAcylated in differentiated CESCs. Finally, O-GlcNAcylation of Sox9 and Runx2 decreased chondrogenesis and increased osteogenesis in CESCs. Conclusions Our findings demonstrate the effect of glucose concentration on regulating the chondrogenic and osteogenic differentiation potential of CESCs and provide insight into the mechanism of how glucose concentration regulates Sox9 and Runx2 O-GlcNAcylation to affect the differentiation of CESCs, which may represent a target for CEP degeneration therapy.

scholarly journals Maintenance of Mitochondrial Morphology by Autophagy and Its Role in High Glucose Effects on Chronological Lifespan ofSaccharomyces cerevisiae

2013 ◽  
Vol 2013 ◽  
pp. 1-13 ◽  
Author(s):  
May T. Aung-Htut ◽  
Yuen T. Lam ◽  
Yu-Leng Lim ◽  
Mark Rinnerthaler ◽  
Cristy L. Gelling ◽  
...  
Keyword(s):  
Conditioned Medium ◽  
High Glucose ◽  
Carbon Sources ◽  
Yeast Cells ◽  
Mitochondrial Morphology ◽  
Glucose Medium ◽  
Mitochondrial Fragmentation ◽  
Chronological Lifespan ◽  
Low Glucose ◽  
Glucose Effects

InSaccharomyces cerevisiae, mitochondrial morphology changes when cells are shifted between nonfermentative and fermentative carbon sources. Here, we show that cells ofS. cerevisiaegrown in different glucose concentrations display different mitochondrial morphologies. The morphology of mitochondria in the cells growing in 0.5% glucose was similar to that of mitochondria in respiring cells. However, the mitochondria of cells growing in higher glucose concentrations (2% and 4%) became fragmented after growth in these media, due to the production of acetic acid; however, the fragmentation was not due to intracellular acidification. From a screen of mutants involved in sensing and utilizing nutrients, cells lackingTOR1had reduced mitochondrial fragmentation, and autophagy was found to be essential for this reduction. Mitochondrial fragmentation in cells grown in high glucose was reversible by transferring them into conditioned medium from a culture grown on 0.5% glucose. Similarly, the chronological lifespan of cells grown in high glucose medium was reduced, and this phenotype could be reversed when cells were transferred to low glucose conditioned medium. These data indicate that chronological lifespan seems correlated with mitochondrial morphology of yeast cells and that both phenotypes can be influenced by factors from conditioned medium of cultures grown in low glucose medium.

scholarly journals Mechanical strain- and high glucose-induced alterations in mesangial cell collagen metabolism: role of TGF-beta.

1998 ◽  
Vol 9 (5) ◽  
pp. 827-836
Author(s):  
B L Riser ◽  
P Cortes ◽  
J Yee ◽  
A K Sharba ◽  
K Asano ◽  
...  
Keyword(s):  
High Glucose ◽  
Collagen Synthesis ◽  
Transforming Growth Factor ◽  
Mechanical Strain ◽  
Collagen Metabolism ◽  
Mrna Levels ◽  
Glucose Medium ◽  
Tgf Beta ◽  
Collagen Accumulation ◽  
Low Glucose

Cultured mesangial cells (MC) exposed to cyclic mechanical strain or high glucose levels increase their secretion of transforming growth factor-beta1 (TGF-beta1) and collagen, suggesting possible mechanisms for the development of diabetic renal sclerosis resulting from intraglomerular hypertension and/or hyperglycemia. This study examines whether glucose interacts with mechanical strain to influence collagen metabolism and whether this change is mediated by TGF-beta. Accordingly, rat MC were grown on flexible-bottom plates in 8 or 35 mM glucose media, subjected to 2 to 5 d of cyclic stretching, and assayed for TGF-beta1 mRNA, TGF-beta1 secretion, and the incorporation of 14C-proline into free or protein-associated hydroxyproline to assess the dynamics of collagen metabolism. Stretching or high glucose exposure increased TGF-beta1 secretion twofold and TGF-beta1 mRNA levels by 30 and 45%, respectively. However, the combination of these stimuli increased secretion greater than fivefold without further elevating mRNA. In 8 mM glucose medium, stretching significantly increased MC collagen synthesis and breakdown, but did not alter accumulation, whereas those stretched in 35 mM glucose markedly increased collagen accumulation. TGF-beta neutralization significantly reduced baseline collagen synthesis, breakdown, and accumulation in low glucose, but had no significant effect on the changes induced by stretch. In contrast, the same treatment of MC in high glucose medium greatly reduced stretch-induced synthesis and breakdown of collagen and totally abolished the increase in collagen accumulation. These results indicate that TGF-beta plays a positive regulatory role in MC collagen synthesis, breakdown, and accumulation. However, in low glucose there is no stretch-induced collagen accumulation, and the effect of TGF-beta is limited to basal collagen turnover. In high glucose media, TGF-beta is a critical mediator of stretch-induced collagen synthesis and catabolism, and, most importantly, its net accumulation. These data have important implications for the pathogenesis and treatment of diabetic glomerulosclerosis.

scholarly journals Glutamate dehydrogenase 1 mediated glutaminolysis sustains HCC cells proliferation and survival under glucose deprivation

2020 ◽  
Author(s):  
Yujiao Zhou ◽  
Yujiao Zhou ◽  
Haibo Yu ◽  
Haibo Yu ◽  
Shengtao Cheng ◽  
...  
Keyword(s):  
Glutamate Dehydrogenase ◽  
High Glucose ◽  
Glucose Deprivation ◽  
Glutamine Metabolism ◽  
Metabolic Flexibility ◽  
Glucose Medium ◽  
Glucose Levels ◽  
Low Glucose ◽  
Glucose Condition ◽  
Hcc Cells

Abstract Background: It is generally believed that tumor cells could sustain its proliferation and survival under different nutrient status according to a so-called metabolic flexibility. How the metabolic flexibility of glutamine metabolism of HCC cells behaves under different glucose conditions has not yet been fully elucidated. In this study, we investigated how the glutamine metabolism modulate the proliferation and survival of HCC cells in response to different glucose conditions and explored the underlying molecular mechanism.Methods: Two cell lines SK-Hep-1 and PLC/PRF/5 were used to evaluate the glutamine addiction of HCC cells. Then, the cells were cultivated in high glucose medium (25mM glucose) and low glucose medium (1.0 mM glucose), respectively, to investigate whether glutaminolysis changed in response to different glucose levels. And, the underlying mechanism of glutamate dehydrogenase 1 (GDH1) sustaining HCC cells survival under glucose deprivation was explored. Additionally, the underlying correlation of GDH1 and glutamate–oxaloacetate transaminase 1 (GOT1) in glucose -poor HCC tissue was investigated.Results: HCC cells were addicted to glutamine. The glutaminolysis of HCC cells was different in response to different glucose conditions. That is, glutamate transaminases GOT1 involved glutamine metabolism played a dominant role in regulating cell growth when glucose was sufficient, while deaminase GDH1 mediated glutaminolysis became dominant when glucose was limited. Mechanically, low-glucose treated HCC cells could induce an elevated expression of GDH1 to supplement the TCA cycles in respond to glucose deprivation. Additionally, we further uncovered an underlying negative association between GDH1 and GOT1 in HCC tissues with decreased glucose levelsConclusions: GDH1 mediated pathway played a leading role in maintaining cell proliferation and survival under low glucose condition. By contrast, GOT1 mediated pathway was activated under high glucose condition. Mechanically, highly expressed GDH1 could drive the TCA cycle in response to glucose deprivation. Besides, there was a potential negative correlation between GDH1 and GOT1 in glucose-poor HCC tissues.

Sign in / Sign up

Export Citation Format

Share Document