Anoxia-tolerant hepatocytes: model system for study of reversible metabolic suppression

1993 ◽  
Vol 265 (1) ◽  
pp. R49-R56 ◽  
Author(s):  
L. T. Buck ◽  
S. C. Land ◽  
P. W. Hochachka
Keyword(s):  
Trypan Blue ◽  
Lactate Production ◽  
Glucose Production ◽  
Isolated Hepatocytes ◽  
Model System ◽  
Blue Staining ◽  
Turnover Rates ◽  
Trypan Blue Exclusion ◽  
Cell Staining ◽  
Chrysemys Picta Bellii

Chrysemys picta bellii is well known for its ability to survive extended anoxic periods and has been widely used as a model system to study anoxic metabolism. Described here is a method for the isolation of anoxia-tolerant hepatocytes from this species. Freshly isolated hepatocytes were determined to be viable based on trypan blue exclusion, gluconeogenic capacity from [14C]lactate, responsiveness to epinephrine and glucagon, and maintenance of cellular adenylate concentrations. Under anoxic conditions for 10 h there was no significant increase in cell staining and no decrease in cellular ATP concentration. Furthermore, the addition of cyanide at the 5-h mark did not result in any significant differences in these parameters; however, iodoacetate added at this time caused trypan blue staining to increase and ATP concentrations to fall. The rate of glucose production from the cells was threefold greater under anoxic than normoxic conditions, underscoring the important role of the liver in supplying substrate during anoxia. From the rate of O2 consumption and rate of lactate production under anaerobic conditions, ATP turnover rates were calculated to be 68.4 +/- 7.2 and 6.5 +/- 0.43 mumol ATP.g-1.h-1, respectively; this corresponds to a 90% decrease in metabolic rate during anoxia. Within a cellular system such as this the more complex regulatory mechanisms involved in a large coordinated reduction in metabolism can be probed.

Protein turnover during metabolic arrest in turtle hepatocytes: role and energy dependence of proteolysis

1994 ◽  
Vol 266 (4) ◽  
pp. C1028-C1036 ◽  
Author(s):  
S. C. Land ◽  
P. W. Hochachka
Keyword(s):  
Energy Dependence ◽  
Protein Turnover ◽  
Lactate Production ◽  
Significant Inhibition ◽  
Turnover Rates ◽  
Atp Turnover ◽  
Protein Pool ◽  
Chrysemys Picta Bellii ◽  
Metabolic Arrest ◽  
Energy Dependent

Hepatocytes from the western painted turtle (Chrysemys picta bellii) are capable of a coordinated metabolic suppression of 88% during 10 h of anoxia at 25 degrees C. The energy dependence and role of proteolysis in this suppression were assessed in labile ([3H]Phe-labeled) and stable ([14C]Phe-labeled) protein pools. During anoxia, labile protein half-lives increased from 24.7 +/- 3.3 to 34.4 +/- 3.7 h, with stable protein half-lives increasing from 55.6 +/- 3.4 to 109.6 +/- 7.4 h. The total anoxic mean proteolytic suppression for both pools was 36%. On the basis of inhibition of O2 consumption and lactate production rates by cycloheximide and emetine, normoxic ATP-dependent proteolysis required 11.1 +/- 1.7 mumol ATP.g-1.h-1 accounting for 21.8 +/- 1.4% of total cellular metabolism. Under anoxia this was suppressed by 93% to 0.73 +/- 0.43 mumol ATP.g-1.h-1. Summation of this with protein synthesis ATP turnover rates indicated that under anoxia 45% of total ATP turnover rate was directed toward protein turnover. Studies with inhibitors of energy metabolism indicated that the majority of energy dependence was found in the stable protein pool, with no significant inhibition occurring among the more labile proteins. We conclude that proteolysis is largely energy dependent under normoxia, whereas under anoxia there is a shift to a slower overall proteolytic rate that is largely energy independent and represents loss mostly from the labile protein pool.

Gluconeogenesis in hepatocytes isolated from the skipjack tuna (Katsuwonus pelamis)

1992 ◽  
Vol 70 (6) ◽  
pp. 1254-1257 ◽  
Author(s):  
L. T. Buck ◽  
R. W. Brill ◽  
P. W. Hochachka
Keyword(s):  
Lactate Dehydrogenase ◽  
Trypan Blue ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Energy Charge ◽  
Lactate Clearance ◽  
Blue Staining ◽  
Skipjack Tuna ◽  
Isolated Cells ◽  
Trypan Blue Exclusion ◽  
Katsuwonus Pelamis

Skipjack tuna (Katsuwonus pelamis) hepatocytes were isolated and determined to be viable based on trypan blue exclusion, energy charge, and lactate dehydrogenase leakage. Trypan blue staining and red blood cell contamination were always less than 0.1%, and lactate dehydrogenase leakage remained less than 1% at 25 °C for 10 h. The energy charge for freshly isolated cells ranged from 0.65 to 0.74. Alanine (10 mM) gave higher rates of gluconeogenesis and oxidation than lactate: alanine rates were 3.17 ± 0.65 μmol glucose/(g packed cells∙h) and 9.1 ± 1.19 μmol CO2/(g∙h), whereas lactate rates were 0.47 ± 0.08 and 1.8 ± 0.27, respectively. Phosphoenolpyruvate carboxykinase was 30% cytosolic and 70% mitochondrial, a finding that is in agreement with the liver's ability to use both lactate and alanine as gluconeogenic substrates. If hepatic gluconeogenesis is the major route of lactate removal postexercise, it is estimated that recovery would require 3800 h! Therefore, tuna liver is not the major site of glucose resynthesis and lactate clearance following burst exercise.

scholarly journals Tissue glucose and lactate metabolism and interconversions in pregnant and lactating sheep

1983 ◽  
Vol 50 (2) ◽  
pp. 267-280 ◽  
Author(s):  
J. G. van der Walt ◽  
G. D. Baird ◽  
E. N. Bergman
Keyword(s):  
Lactate Production ◽  
Blood Flow Rate ◽  
Glucose Production ◽  
Late Pregnancy ◽  
Whole Body ◽  
Early Lactation ◽  
Lactate Metabolism ◽  
Turnover Rates ◽  
Peripheral Tissues ◽  
Pregnancy And Lactation

1. Continuous infusions of [14C]glucose and [14C]lactate on separate days, and measurements of blood flow-rate, were used to obtain values for rates of unidirectional metabolism and of interconversion of glucose and lactate in the portal-drained viscera, liver and hind-quarters of ewes during late pregnancy and early lactation. All infusions were made within 5 h after the morning meal, when steady-state conditions appeared to exist.2. Use was made of ewes that had been appropriately catherized during pregnancy, and whose catheter remained patent through into lactation.3. The liver was the main source of glucose production (67–70%) during both pregnancy and lactation. Other sources were the portal-drained viscera (absorbed glucose) and, presumably, the kidneys. Over 80% of the glucose was utilized by the peripheral tissues with approximately 35–40% of utilization being attributable to the hind-quarters.4. Of the total lactate production, 76% occurred in the peripheral tissues during pregnancy but only 36% during lactation. While the liver utilized 73% of lactate during pregnancy, this value fell to only 42% during lactation, at which time the portal-drained viscera utilized 26% of the lactate.5. During pregnancy, approximately 80% of the lactate arose from glucose, chiefly in peripheral tissues, while at least 12% of the glucose arose from lactate, chiefly in the liver. During lactation the extent of these interconversions was decreased.6. Despite the interconversions, whole-body turnover rates for glucose and lactate were under- or overestimated by only 4–10% and 2–5% respectively. Furthermore, a comparison of turnover rates obtained with [U-14C]- and [6−3H]glucose indicated that there was only 6 and 2% recycling of glucose-C during pregnancy and lactation respectively.7. Under the conditions employed in this study, lactate does not appear to be a major precursor of glucose in the ruminant, and most of the lactate taken up by the liver must be used for purposes other than gluconeogenesis, such as oxidation or alternative anabolic pathways.

Microcalorimetric measurement of reversible metabolic suppression induced by anoxia in isolated hepatocytes

1993 ◽  
Vol 265 (5) ◽  
pp. R1014-R1019 ◽  
Author(s):  
L. T. Buck ◽  
P. W. Hochachka ◽  
A. Schon ◽  
E. Gnaiger
Keyword(s):  
Heat Flux ◽  
Lactate Production ◽  
Isolated Hepatocytes ◽  
Enthalpy Change ◽  
Short Term ◽  
G Cells ◽  
Chrysemys Picta Bellii ◽  
Metabolic Suppression ◽  
Free Glucose ◽  
Normoxic Control

The metabolic suppression due to anoxia in hepatocytes from the anoxia-tolerant turtle Chrysemys picta bellii was measured directly using microcalorimetric techniques. The normoxic heat flux from hepatocytes in suspension (25 degrees C) was 1.08 +/- 0.08 mW/g cells and decreased by 76% to 0.26 +/- 0.03 mW/g cells in response to anoxic incubation. After an acute decrease in temperature (to 10 degrees C) anoxic heat flux dropped by 96% relative to the normoxic control at 25 degrees C. The relative decrease in heat flux at both temperatures was similar, 76% at 25 degrees C and 68% at 10 degrees C. From the caloric equivalent of glycogen fermentation to lactate the heat flux from lactate production was calculated to be -93 microW/g cells (25 degrees C), and this accounted for 36% of the anoxic heat flux. When the enthalpy change associated with the release of free glucose (from glycogen breakdown) is considered, an additional 6% of the anoxic heat flux can be accounted for. Therefore, a portion of the anoxic heat flux is unaccounted for (58%), resulting in an “exothermic gap.” This differs from the normoxically incubated hepatocytes where the indirect calorimetric measurement of heat flux (hepatocyte O2 consumption) could fully account for the calorimetrically measured heat flux. When normoxic hepatocytes were inhibited with cyanide, a rapid suppression in heat flux was observed. Because rapid reequilibration to a lower, cyanide-induced steady state occurred in < 15 min, it is also assumed that there is no short-term Pasteur effect in this tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

Polydopamine and dopamine interfere with tetrazolium-based cytotoxicity assays and produce exaggerated cytocompatibility inferences

2021 ◽  
Author(s):  
Anand Kumar Awasthi ◽  
Sakshi Gupta ◽  
Kavthekar Rupesh Namdev ◽  
Aditi Banerjee ◽  
Aasheesh Srivastava
Keyword(s):  
Cell Viability ◽  
Trypan Blue ◽  
Reliable Estimate ◽  
Trypan Blue Exclusion ◽  
Cytotoxicity Assays ◽  
Trypan Blue Exclusion Assay

Polydopamine (PDA) and dopamine (DA) can spontaneously reduce MTT reagent to formazan, resulting in incorrect cell-viability inferences. The non-redox Trypan Blue exclusion assay provides a more reliable estimate of cell viability with PDA and DA.

Trypan blue staining in vitreoretinal surgery

Ophthalmology ◽  
2004 ◽  
Vol 111 (8) ◽  
pp. 1622-1623 ◽  
Author(s):  
Gema Rebolleda ◽  
Francisco José Muñoz Negrete ◽  
Marta Suarez-Figueroa
Keyword(s):  
Trypan Blue ◽  
Vitreoretinal Surgery ◽  
Blue Staining ◽  
Trypan Blue Staining

Use of trypan blue staining to assess the quality of ovarian cryopreservation

2007 ◽  
Vol 87 (5) ◽  
pp. 1200-1207 ◽  
Author(s):  
Patricia Fauque ◽  
Anis Ben Amor ◽  
Christiane Joanne ◽  
Germain Agnani ◽  
Jean Luc Bresson ◽  
...  
Keyword(s):  
Trypan Blue ◽  
Blue Staining ◽  
Trypan Blue Staining

scholarly journals PHYTOCHEMICAL STUDY AND THE ANTIPROLIFERATIVE ACTIVITY OF INULA VULGARIS SPECIES GROWN IN LEBANON

2017 ◽  
Vol 9 (8) ◽  
pp. 75
Author(s):  
Assi M. ◽  
Usta J. ◽  
Mounimne Y. ◽  
Aboul Ela M. ◽  
El Lakany A.
Keyword(s):  
Ethyl Acetate ◽  
Antiproliferative Activity ◽  
Trypan Blue ◽  
Heart Diseases ◽  
Trypan Blue Exclusion ◽  
Potent Effect ◽  
Phytochemical Study ◽  
Chemical Structures ◽  
13C Nuclear Magnetic Resonance ◽  
Mtt Assays

Objective: Cancer represents the second leading cause of death after stroke and heart diseases. Plant extracts have long been used in traditional medicine for the prevention and treatment of many illnesses, including some types of cancer. The aim of this study was to evaluate the antiproliferative effects of ethyl acetate fractions of two Lebanese herbs: Inula viscosa (I. vis) and Inula vulgaris (I. vul).Methods: Plants were extracted with ethanol followed by ethyl acetate, then dried and tested on three cell lines including CaCO2, HepG2, and MCF7, to check for their viability and antiproliferative activity, using trypan blue exclusion and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Proton (1H) and carbon (13C) nuclear magnetic resonance spectrometry (NMR) were used to identify the compounds that have been isolated from both Inula species.Results: The current findings were consistent for both trypan blue and MTT assays. The results showed that the most potent effect for I. vul was HepG2 (IC50 20 µg/ml, 27 µg/ml), and for I. vis on MCF7 (9 µg/ml, 15 µg/ml) and CaCO2 (12 µg/ml, 22 µg/ml) in the two mentioned assays respectively. However, insignificant differences were observed among the studied plants for each of the evaluated cells indicating comparable potencies. Quercetin, quercetin glycoside, and epicatechin derivatives were isolated by fractionation on column chromatography and identified using NMR spectroscopy.Conclusion: The antiproliferative activities of the two plants could be related to their content that is significant for high levels of secondary metabolites. The identification of those compounds is necessary to establish a relationship between their chemical structures and their activities.

Effect of a mixed meal on hepatic lactate and gluconeogenic precursor metabolism in dogs

1984 ◽  
Vol 247 (3) ◽  
pp. E362-E369 ◽  
Author(s):  
M. A. Davis ◽  
P. E. Williams ◽  
A. D. Cherrington
Keyword(s):  
Hepatic Glucose Production ◽  
Lactate Production ◽  
Glucose Production ◽  
Hepatic Uptake ◽  
Mixed Meal ◽  
Arterial Lactate ◽  
Hepatic Glucose ◽  
Hepatic Glucose Uptake ◽  
Arterial Glucose ◽  
Arterial Lactate Level

The present experiments were undertaken to assess lactate and gluconeogenic precursor metabolism in the 30 h following consumption of a mixed meal by the overnight-fasted, conscious dog. The arterial glucose level rose by a maximum of 13 mg/dl 4 h after the meal and had returned to control levels by 12 h. Hepatic glucose production was suppressed for 12 h after feeding, but net hepatic glucose uptake did not occur. The arterial lactate level rose from 0.55 +/- 0.10 to 1.28 +/- 0.14 mM within 1 h of feeding and remained elevated for 12 h. Net hepatic lactate production, measured with an A-V difference technique, rose from 3.5 +/- 2.8 to 19.4 +/- 3.1 mumol X kg-1 X min-1 h after the meal and declined slowly over the next 22 h. The liver then began to consume lactate so that at 30 h net hepatic uptake was 5.7 +/- 0.5 mumol X kg-1 X min-1. The total hepatic uptake of the gluconeogenic amino acids (alanine, glycine, serine, threonine) increased from 5.3 +/- 0.8 to 11.5 +/- 2.5 mumol X kg-1 X min-1 at 1 h and remained elevated for 4 h. The arterial alanine level rose from 0.36 +/- 0.03 to 0.51 +/- 0.04 mM at 2 h and remained elevated for 18 h. Insulin increased from 11 +/- 2 microU/ml to a maximum of 44 +/- 5 4 h after the meal, and the glucagon level rose from 59 +/- 8 pg/ml to a maximum of 150 +/- 22 1 h after feeding.(ABSTRACT TRUNCATED AT 250 WORDS)

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