Differential effects of respiratory inhibitors on glycolysis in proximal tubules

1990 ◽  
Vol 258 (6) ◽  
pp. F1608-F1615 ◽  
Author(s):  
K. G. Dickman ◽  
L. J. Mandel
Keyword(s):  
Oxidative Metabolism ◽  
Cell Injury ◽  
Lactate Production ◽  
Proximal Tubules ◽  
Antimycin A ◽  
Transport Function ◽  
Atp Production ◽  
Rotenone Treatment ◽  
Glycolytic Atp ◽  
Ldh Release

The effects of inhibition of mitochondrial energy production at various points along the respiratory chain on glycolytic lactate production and transport function were examined in a suspension of purified rabbit renal proximal tubules. Paradoxically, partial blockage at site 3 by hypoxia (1% O2) induced lactate production, whereas total site 3 blockage by anoxia (0% O2) failed to stimulate glycolysis. Compared with anoxia, hypoxic tubules exhibited greater preservation of ATP and K+ contents during O2 deprivation and more fully recovered oxidative metabolism and transport function during reoxygenation. The mitochondrial site 1 inhibitor rotenone and the uncoupler carbonyl cyanide-p-trifluorome-thoxyphenylhydrazone (FCCP) were equipotent stimuli for lactate production, whereas the site 2 inhibitor antimycin A failed to stimulate glycolysis despite a 90% inhibition of O2 consumption. Compared with antimycin A, treatment with rotenone or FCCP resulted in less cell injury [measured by lactate dehydrogenase (LDH) release] and greater preservation of cell K+ and ATP contents. 2-Deoxyglucose blocked lactate production by 50% in the presence of rotenone and increased LDH release, suggesting that glycolytic ATP is partially protective. Addition of ouabain during rotenone treatment reduced lactate production by 50%, indicating that glycolytic ATP can be used to fuel the Na pump when mitochondrial ATP production is inhibited. We conclude that 1) proximal tubules can generate lactate during inhibition of oxidative metabolism by hypoxia, rotenone, or FCCP; 2) mitochondrial inhibition is not obligatorily linked to activation of glycolysis, since neither anoxia nor antimycin A stimulate lactate production; 3) when ATP can be produced through anaerobic glycolysis it serves to protect cell viability and transport function during respiratory inhibition.

Ca2+ uptake, fatty acid, and LDH release during proximal tubule hypoxia: effects of mepacrine and dibucaine

1994 ◽  
Vol 266 (2) ◽  
pp. F196-F201 ◽  
Author(s):  
D. Bunnachak ◽  
A. R. Almeida ◽  
J. F. Wetzels ◽  
P. Gengaro ◽  
R. A. Nemenoff ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Lactate Dehydrogenase ◽  
Proximal Tubule ◽  
Uptake Rate ◽  
Cell Injury ◽  
Potential Effect ◽  
Proximal Tubules ◽  
Membrane Injury ◽  
Ldh Release ◽  
Higher Doses

In freshly isolated hypoxic rat proximal tubules, Ca2+ uptake rate increases promptly, within 1 min, and remains significantly elevated throughout a 20-min period of hypoxia. Lactate dehydrogenase (LDH) release, a sign of membrane injury, increases only after 5 min of hypoxia and thereafter rises progressively. The potential effect of increased Ca2+ uptake rate to activate phospholipases, which would then initiate membrane injury, was evaluated by treating hypoxic tubules with three dissimilar phospholipase inhibitors, i.e., mepacrine, dibucaine, or p-bromophenacyl bromide (PBPB). LDH release averaged 11.9 and 13.8% after 10 and 20 min of normoxia, respectively. With 10 or 20 min of hypoxia LDH release increased to 46.0 and 65.2%, respectively (P < 0.01), and Ca2+ uptake rate increased from 2.56 in normoxia to 4.71 nmol.mg-1 x min-1 at 10 min of hypoxia (P < 0.01) and from 2.82 in normoxia to 3.76 nmol/mg at 20 min of hypoxia (P < 0.05). In a separate series of tubules, after 10 min of hypoxia LDH release was reduced by pretreatment with 50 microM mepacrine (66.1 to 47.3%, P < 0.01) or 50 microM dibucaine (53.1 to 38.5%, P < 0.02). The increase in Ca2+ uptake rate also was significantly reduced. After 20 min of hypoxia neither mepacrine nor dibucaine reduced Ca2+ uptake rate; LDH release was modestly reduced by dibucaine but not mepacrine. Higher doses of mepacrine (500 microM) and dibucaine (250 microM) also reduced cell injury at 10 min of hypoxia as assessed by LDH release.(ABSTRACT TRUNCATED AT 250 WORDS)

Cultivation in Glucose-Deprived Medium Stimulates Mitochondrial Biogenesis and Oxidative Metabolism in HepG2 Hepatoma Cells

2002 ◽  
Vol 383 (2) ◽  
pp. 283-290 ◽  
Author(s):  
K. Weber ◽  
D. Ridderskamp ◽  
M. Alfert ◽  
S. Hoyer ◽  
R.J. Wiesner
Keyword(s):  
Mitochondrial Dna ◽  
Mitochondrial Biogenesis ◽  
Oxidative Metabolism ◽  
Hepg2 Cells ◽  
Energy Supply ◽  
Energy Requirement ◽  
Lactate Production ◽  
Fold Increase ◽  
Atp Production ◽  
Mitochondrial Transcription Factor

Abstract In order to test the hypothesis that an imbalance between energy requirement and energy supply regulates mitochondrial genes and ultimately mitochondrial biogenesis, energy supply was challenged in HepG2 cells by withdrawal of glucose from the culture medium, making the cells exclusively dependent on mitochondrial ATP production. Such cells showed a 2-fold increase of cytochrome c oxidase activity, elevated levels of mitochondrial DNA, mitochondrial DNA encoded mRNAs and proteins, as well as the nuclear encoded mitochondrial transcription factor A. Lactate production was significantly reduced and glutamine was consumed as an alternative substrate for oxidative metabolism. Longterm adapted cells formed exclusively monolayers, while they normally grow in multilayers forming tumor spheroids. Also, longterm adapted cells proliferated significantly faster. No differences for the ATP/ADP ratio were observed, indicating that this is not the primary signal initiating the adaptative processes. These results show that mitochondrial biogenesis and oxidative metabolism are stimulated in HepG2 cells grown in the absence of fermentable glucose, probably in order to compensate for the diminished supply of glycolytic ATP.

Lactate production in isolated segments of the rat nephron

1985 ◽  
Vol 248 (4) ◽  
pp. F522-F526 ◽  
Author(s):  
S. Bagnasco ◽  
D. Good ◽  
R. Balaban ◽  
M. Burg
Keyword(s):  
Collecting Duct ◽  
Lactate Production ◽  
Proximal Tubules ◽  
Antimycin A ◽  
Anaerobic Glycolysis ◽  
Metabolic Substrate ◽  
Collecting Ducts ◽  
Medullary Collecting Duct ◽  
Convoluted Tubules

Lactate production was measured directly in individual segments of the rat nephron. Tubules were dissected and then incubated in vitro with glucose as the only metabolic substrate. Each segment was incubated with and without antimycin A, an inhibitor of oxidative metabolism. Proximal tubules produced no lactate with or without antimycin A. The distal segments all produced lactate. The rate of lactate production without antimycin A ranged from 0.4 to 0.9 pmol X min-1 X mm-1 in all distal segments except one, the inner medullary collecting duct, which produced lactate at the significantly higher rate of 2.8 pmol X min-1 X mm-1. Antimycin A increased lactate production significantly in all of the distal segments. The increase was largest in medullary thick ascending limbs (1,400%) and cortical (798%) and outer medullary collecting ducts (357%). Increments were smaller in cortical thick ascending limbs (98%) and distal convoluted tubules (98%) and least in the inner medullary collecting ducts (28%). We conclude that lactate production occurs only in distal segments of the nephron and that under anoxic conditions significant amounts of ATP are produced by anaerobic glycolysis in these segments.

Metabolic inhibitors: effects on metabolism and transport in the proximal tubule

1982 ◽  
Vol 243 (2) ◽  
pp. F133-F140 ◽  
Author(s):  
S. R. Gullans ◽  
P. C. Brazy ◽  
S. P. Soltoff ◽  
V. W. Dennis ◽  
L. J. Mandel
Keyword(s):  
Oxidative Metabolism ◽  
Metabolic Inhibitors ◽  
Antimycin A ◽  
Atp Content ◽  
Atp Production ◽  
Sodium Dependent ◽  
Dependent Transport ◽  
Convoluted Tubules ◽  
Mitochondrial Oxidative Metabolism ◽  
Made In

This study examined the effects of the metabolic inhibitors rotenone and antimycin A on oxidative metabolism and transport in the proximal renal tubule of the rabbit. Measurements of oxygen consumption rate (QO2), cellular ATP content, and mitochondrial NAD redox state were made in suspensions of renal cortical tubules. Parallel experiments were conducted in isolated perfused proximal convoluted tubules to measure the absorption rates of fluid (Jv), phosphate (JlbPO4), and glucose (JlbGlc). The results indicate that rotenone and antimycin A, at doses (10(-6) M) that maximally inhibited mitochondrial oxidative phosphorylation, abolished net fluid and phosphate absorption and nearly eliminated net glucose transport. Partial inhibition of oxidative metabolism with 10(-7) M rotenone caused proportional reductions in QO2, ATP content, and Jv; however, JlbPO4 was reduced more markedly than either Jv or JlbGlc. We conclude that rotenone and antimycin A inhibit the sodium-dependent transport of fluid, phosphate, and glucose by blocking mitochondrial ATP production. Furthermore, the inhibition of mitochondrial oxidative metabolism and the inhibition of net sodium transport are closely correlated.

Effects of Antimycin A and 2-Deoxyglucose on Energy Metabolism in Washed Human Platelets

1979 ◽  
Vol 42 (05) ◽  
pp. 1460-1472 ◽  
Author(s):  
Holm Holmsen ◽  
Linda M Robkin
Keyword(s):  
Plasma Proteins ◽  
Platelet Rich Plasma ◽  
Energy Charge ◽  
Lactate Production ◽  
Human Platelets ◽  
Metabolic Inhibitors ◽  
Antimycin A ◽  
Adenylate Energy Charge ◽  
Atp Production ◽  
Major Mechanism

Summary(1) Human platelets were incubated with [14C]adenine in plasma, washed and resuspended in salt solutions. The effects of incubating the cells with antimycin A and 2- deoxyglucose on the radioactivity of ATP, ADP, AMP, IMP and inosine + hypoxanthine, the total level of ADP and ATP and lactate production were studied. The metabolic inhibitors only affected the cytoplasmic, [14C]-labelled nucleotides, and were apparently without effect on the granular, non-labelled ATP and ADP. (2) Antimycin A caused a rapid, shortlasting decrease in [14C]ATP which was independent on glucose and enhanced by 2- deoxyglucose. Lactate production increased about 3-fold with and without 2-deoxyglucose. The initial fall in [14C]ATP was therefore thought to be due to a failure to immediately substitute for the lost oxidative ATP production. (3) After the initial fall in [14C]ATP no changes took place when glucose was present, while [14C]ATP and the adenylate energy charge decreased when glucose was absent and more so when 2-deoxyglucose was present. Thus, glycogenolysis, corresponding to an ATP turnover of 29.8 nmoles × min-1 × mg-1 protein did not maintain ATP homeostasis in platelets. (4) The changes in [14C]ATP, adenylate energy charge and lactate production under the various conditions strongly suggested that regulation of ATP consumption in the cells was a major mechanism to maintain ATP homeostasis. (5) The changes in the [14C]nucleotides in washed platelets were qualitatively similar to those previously described in platelet-rich plasma (Holmsen et al. 1974); quantitative differences were apparent and found to be due to binding of antimycin A to plasma proteins and counteraction of the effects of 2-deoxyglucose by plasma glucose. In particular, the rapid, initial fall in [14C]ATP occurred only with washed cells and clearly demonstrated a conversion of ATP to hypoxanthine ADP, AMP, IMP and inosine. (6) The cellular AMP deaminase reaction was found not to be related by the adenylate energy charge, in contrast to studies with semipurified enzyme (Chapman & Atkinson 1973). Our results suggested that AMP deamination occurred when [AMP] rose from its resting value of 0.07 mM and was inhibited when [ATP] fell below 2 mM within the cell.

Protection by glycine of proximal tubules from injury due to inhibitors of mitochondrial ATP production

1990 ◽  
Vol 258 (6) ◽  
pp. C1127-C1140 ◽  
Author(s):  
J. M. Weinberg ◽  
J. A. Davis ◽  
M. Abarzua ◽  
T. Kiani ◽  
R. Kunkel
Keyword(s):  
Mitochondrial Function ◽  
Structural Integrity ◽  
Cell Injury ◽  
Buthionine Sulfoximine ◽  
Proximal Tubules ◽  
Atp Depletion ◽  
Protective Effects ◽  
Atp Production ◽  
Carbonyl Cyanide ◽  
Severe Impairment

We have determined whether glycine or glutathione can protect rabbit proximal tubules damaged by chemical inhibitors of oxidative phosphorylation: antimycin A, rotenone, cyanide, oligomycin, or carbonyl cyanide m-chlorophenylhdrazone (CCCP). All the agents severely depleted cell ATP levels within 15 min and caused lethal cell injury, as quantified by lactate dehydrogenase (LDH) release. Glycine and glutathione largely prevented this injury without altering the primary effects of the inhibitors on tubule respiration or the depletion of ATP. Buthionine sulfoximine and 1,3-bis(2-chloroethyl)-1-nitrosourea decreased cell glutathione but did not prevent the protective effects of either glycine or glutathione in tubules treated with rotenone. Protection was sustained during both a 15-min exposure and a 45-min postwash period irrespective of whether the wash removed the agent or mitochondrial function recovered. Cysteine uniquely induced a dramatic recovery of mitochondrial function in tubules washed after treatment with CCCP. These data 1) demonstrate that the cytoprotective effects of glycine previously seen during hypoxia extend to other tubule lesions characterized by severe ATP depletion, 2) emphasize the actions of glycine to preserve cell structural integrity in spite of sustained severe impairment of ATP-generating processes in proximal tubules, and 3) indicate that it is glycine rather than intracellular or extracellular glutathione which mediates protection.

Absence of endonuclease activation during acute cell death in renal proximal tubules

1993 ◽  
Vol 265 (2) ◽  
pp. C485-C490 ◽  
Author(s):  
R. G. Schnellmann ◽  
A. R. Swagler ◽  
M. M. Compton
Keyword(s):  
Cell Death ◽  
Proximal Tubule ◽  
Calcium Ionophore ◽  
Renal Proximal Tubule ◽  
Proximal Tubules ◽  
Free Calcium ◽  
Proximal Tubule Cell ◽  
Antimycin A ◽  
Aurintricarboxylic Acid ◽  
Ldh Release

The role of endonuclease and poly(ADP-ribose) polymerase activation in various types of cell injuries and death to rabbit renal proximal tubule suspensions was examined. Proximal tubules were exposed to the mitochondrial inhibitor antimycin A (0.1 microM), the protonophore carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP, 1 microM), the calcium ionophore ionomycin (5 microM), or the oxidant t-butyl hydroperoxide (TBHP, 0.5 mM) in the absence or presence of the endonuclease inhibitor aurintricarboxylic acid or the poly(ADP-ribose) polymerase inhibitor 3-aminobenzamide. Lactate dehydrogenase (LDH) release was used as a marker of cell death and analysis of genomic DNA for internucleosomal cleavage was used as a marker of endonuclease activation. Aurintricarboxylic acid and 3-aminobenzamide had no effect on the proximal tubule LDH release produced by 1 h exposure to antimycin A, FCCP, or ionomycin, or 2 h exposure to TBHP. Furthermore, there was no evidence of DNA fragmentation with any compound prior to or after cell death began. As a positive control, proximal tubules exposed to digitonin in the absence of metabolic substrates resulted in the chelator-inhibitable fragmentation of DNA, indicating that the endonuclease is present in proximal tubules. These results show that endonuclease activation did not occur prior to or after cell death began. Furthermore, these results suggest that endonuclease and poly(ADP-ribose) polymerase activation do not play a role in this model of acute renal proximal tubule cell injury and death induced by agents that cause oxidative stress, mitochondrial dysfunction, or increases in cytosolic free calcium.

Carbohydrate metabolism by primary cultures of rabbit proximal tubules

1989 ◽  
Vol 256 (3) ◽  
pp. C532-C539 ◽  
Author(s):  
M. J. Tang ◽  
K. R. Suresh ◽  
R. L. Tannen
Keyword(s):  
Pyruvate Kinase ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Cultured Cells ◽  
Primary Cultures ◽  
Lactate Production ◽  
Proximal Tubules ◽  
Glycolytic Metabolism ◽  
Proximal Tubular Cells ◽  
Tubular Cells ◽  
Proximal Tubular

Renal proximal tubular epithelia were used to assess the factors responsible for the induction of glycolysis in cultured cells. Primary cultures of rabbit proximal tubules, which achieved confluency at 6 days, exhibited hormonal responsiveness and brush-border characteristics typical of proximal tubular cells. Beginning at day 4, these cultured cells exhibited increased glycolytic metabolism reflected by enhanced glucose uptake and lactate production, along with parallel increases in activity of the glycolytic enzymes, pyruvate kinase and lactate dehydrogenase. The gluconeogenic enzymes, phosphoenolpyruvate carboxykinase (PEPCK) and fructose-1,6-bisphosphatase (FDP), were downregulated, and the cultured cells exhibited lower oxygen consumption rates than fresh tubules. Cells grown on a rocker, to mitigate hypoxia, exhibited a metabolic and enzymatic profile similar to cells grown under still conditions. ATP levels in cultured cells were higher than in fresh tubules. Furthermore, pyruvate kinase activity was higher in cells grown in media containing 0.5 as contrasted with 25 mM glucose. The enhanced glycolytic metabolism exhibited by cultured proximal tubular cells appears to be a characteristic of proliferation and is not a response to hypoxia, the Pasteur effect, or environmental glucose.

Reduced high-energy phosphate levels in rat hearts. I. Effects of alloxan diabetes

1976 ◽  
Vol 230 (6) ◽  
pp. 1744-1750 ◽  
Author(s):  
TB Allison ◽  
SP Bruttig ◽  
Crass MF ◽  
RS Eliot ◽  
JC Shipp
Keyword(s):  
Oxidative Metabolism ◽  
Oxygen Delivery ◽  
Rat Heart ◽  
High Energy ◽  
Diabetic Rat ◽  
High Energy Phosphate ◽  
Atp Production ◽  
Rat Hearts

Significant alterations in heart carbohydrate and lipid metabolism are present 48 h after intravenous injection of alloxan (60 mg/kg) in rats. It has been suggested that uncoupling of oxidative phosphorylation occurs in the alloxanized rat heart in vivo, whereas normal oxidative metabolism has been demonstrated in alloxan-diabetic rat hearts perfused in vitro under conditions of adequate oxygen delivery. We examined the hypothesis that high-energy phosphate metabolism might be adversely affected in the alloxan-diabetic rat heart in vivo. Phosphocreatine and ATP were reduced by 58 and 45%, respectively (P is less than 0.001). Also, oxygen-dissociation curves were shifted to the left by 4 mmHg, and the rate of oxygen release from blood was reduced by 21% (P is less than 0.01). Insulin administration normalized heart high-energy phosphate compounds. ATP production was accelerated in diabetic hearts perfused in vitro with a well-oxygenated buffer. These studies support the hypothesis that oxidative ATP production in the alloxan-diabetic rat heart is reduced and suggest that decreased oxygen delivery may have a regulatory role in the oxidative metabolism of the diabetic rat heart.

scholarly journals MiR-195-5p Reduces Esophageal Cancer Cell Proliferation Through The IGF-1R/AKT Axis

2021 ◽  
Author(s):  
Zhao-xian Lin ◽  
Xing Lin ◽  
Lihuan Zhu ◽  
Jian-yuan Huang ◽  
Yang-yun Huang
Keyword(s):  
Esophageal Cancer ◽  
Lactate Production ◽  
Cancer Cell Proliferation ◽  
Poor Survival ◽  
Atp Production ◽  
Downstream Target ◽  
Glut1 Expression ◽  
Esophageal Cancer Cell ◽  
Low Levels ◽  
Novel Target

Abstract Background Esophageal cancer (ECa) remains a major cause of mortality across the globe. The expression of MiR-195-5p is altered in a plethora of tumors, but its role in ECa development and progression are undefined. Result Here, we show that miR-195-5p is downregulated in ECa and associated with poor survival in ECa. Function assays indicated that MiR-195-5p inhibited ECa progression. Mechanistically, we identified IGF-1R as a downstream target of miR-195-5p, and miR-195-5p/IGFR axis caused a loss of GLUT1 expression, reduced glucose uptake, reduced lactate production, and low levels of ATP production. Conclusion Collectively, miR-195-5p as a Eca suppressor impaired glycolysis. This highlighting miR-195-5p as a novel target for much needed anti-ECa therapeutics.

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