scholarly journals Stimulation of mitochondrial pyruvate metabolism and citrulline synthesis by dexamethasone. Effect of isolation and incubation media

1984 ◽  
Vol 222 (2) ◽  
pp. 379-387 ◽  
Author(s):  
A D Martin ◽  
M A Titheradge
Keyword(s):  
Adenine Nucleotides ◽  
Isolation Procedure ◽  
Mitochondrial Matrix ◽  
Pyruvate Metabolism ◽  
Pyruvate Carboxylation ◽  
Matrix Volume ◽  
The Matrix ◽  
Citrulline Synthesis ◽  
Stimulation Of

Hepatic mitochondria isolated in 0.3 M-sucrose or 0.3 M-mannitol from rats treated for 3h with dexamethasone displayed stimulated rates of pyruvate carboxylation and decarboxylation and citrulline synthesis when compared with organelles from control animals. Mitochondria isolated in mannitol also displayed elevated rates of pyruvate carboxylation and decarboxylation when compared with those isolated in sucrose, and this stimulation was shown to be independent of the lengthy isolation procedure. Citrulline synthesis proceeded at similar rates in mitochondria isolated in either sugar. The concentration of exchangeable adenine nucleotides was identical in mitochondria isolated in sucrose or mannitol, suggesting that those prepared in the former sugar are not more permeable to metabolites than those prepared in the latter. The matrix volume of mitochondria isolated in mannitol was greater than that of mitochondria isolated in sucrose, and the effect of mannitol on pyruvate metabolism was mimicked by swelling the organelles in hypo-osmotic sucrose. Measurements of the extra-matrix volume by using [14C]sucrose or [14C]mannitol suggest that mannitol can permeate mitochondria to a greater extent than can sucrose. The possibility that mannitol elicits its effect by entering the mitochondrial matrix and so initiating swelling is discussed.

scholarly journals Stimulation of mitochondrial functions by glucagon treatment. Evidence that effects are not artifacts of mitochondrial isolation

1983 ◽  
Vol 210 (3) ◽  
pp. 819-827 ◽  
Author(s):  
C B Jensen ◽  
F D Sistare ◽  
H C Hamman ◽  
R C Haynes
Keyword(s):  
Phosphoenolpyruvate Carboxykinase ◽  
Treatment Result ◽  
Direct Result ◽  
Differential Centrifugation ◽  
Hormonal Stimulation ◽  
Pyruvate Carboxylation ◽  
Mitochondrial Functions ◽  
Liver Homogenates ◽  
Citrulline Synthesis ◽  
Stimulation Of

(1) Activation of rat liver mitochondrial functions following glucagon treatment was demonstrated in mitochondria that had not been isolated by the conventional technique of differential centrifugation and washing in sucrose solutions. Crude liver homogenates in 0.3 M-sucrose or 0.15 M-KCl prepared from rats treated with glucagon showed stimulation of State-3 and uncoupled respiration, carboxylation of pyruvate, and citrulline synthesis comparable with those previously reported in isolated mitochondria. (2) During the isolation procedure of mitochondria the hormonal stimulations of pyruvate carboxylation and citrulline formation were shown not to be enhanced by sequential washing. (3) Mitochondria isolated from glucagon-treated rats by differential centrifugation and washing in 0.3 M-mannitol/1 mM-EGTA, pH 7.0, exhibited a mean rate of citrulline synthesis that was greater than twice that of the control. Liver homogenates prepared in 0.3 M-sucrose or 0.3 M-mannitol showed identical rates of State-3 respiration and percentage stimulations of respiration by glucagon treatment. (4) Addition of glucagon led to a rapid accumulation of malate and aspartate and decreased the amounts of glutamate and citrate in isolated hepatocytes incubated with L-lactate. When gluconeogenesis was inhibited at the phosphoenolpyruvate carboxykinase (EC 4.1.1.32) reaction these phenomena were accentuated, lending support to the interpretation that they are the direct result of stimulation of carboxylation and oxidation reactions in the mitochondria. These results do not support the proposal [Siess, Fahimi & Wieland (1981) Hoppe-Seyler's Z. Physiol. Chem. 362. 1643-1651] that the mitochondrial effects of glucagon treatment result from a stabilization of mitochondria to detrimental effects of sucrose during their isolation. (5) The mean hormonal stimulation of pyruvate carboxylation in mitochondria isolated in 0.3 M-sucrose was shown to be approx. 2.5-fold when assayed either at 37 degrees C or 25 degrees C. In contrast, on the basis of similar experiments, Siess et al. (1981) concluded that the effects of glucagon on hepatic mitochondria are not characteristic of a true hormonal stimulation. Our data indicate this conclusion to be unjustified.

scholarly journals Rat liver mitochondria prepared in mannitol media demonstrate increased mitochondrial volumes compared with mitochondria prepared in sucrose media. Relationship to the effect of glucagon on mitochondrial function

1984 ◽  
Vol 221 (1) ◽  
pp. 147-152 ◽  
Author(s):  
D E Whipps ◽  
A P Halestrap
Keyword(s):  
Liver Mitochondria ◽  
Mitochondrial Metabolism ◽  
Rat Liver Mitochondria ◽  
Pyruvate Metabolism ◽  
Succinate Oxidation ◽  
Isolation Medium ◽  
Matrix Volume ◽  
Sucrose Medium ◽  
Mitochondrial Volume ◽  
Citrulline Synthesis

Liver mitochondria isolated from glucagon-treated rats by using both mannitol- and sucrose-based media showed enhanced uncoupled succinate oxidation, pyruvate metabolism and citrulline synthesis. Mitochondria prepared in mannitol medium showed some stimulation of these parameters compared with those prepared in sucrose medium. This was accompanied by an increase in matrix volume of about 20%. Some [14C]mannitol became permanently associated with mitochondria during preparation. It is suggested that mannitol may enter mitochondria during their preparation and cause swelling. The presence of 4mM-phosphate in the sucrose isolation medium stimulated the same parameters as did glucagon treatment, and also caused an increase in matrix volume of about 20%. These results confirm the conclusion that the mitochondrial volume may be important in the regulation of mitochondrial metabolism. They contradict the conclusion of others [Siess (1983) Hoppe-Seyler's Z. Physiol. Chem. 364, 279-290, 835-838] that mannitol rather than sucrose should be used when studying hormonal effects on mitochondrial metabolism. Reasons for the discrepancies in the results between groups studying the effects of hormones on mitochondrial metabolism are discussed.

scholarly journals The stimulation of mitochondrial pyruvate carboxylation after dexamethasone treatment of rats

1984 ◽  
Vol 219 (1) ◽  
pp. 107-115 ◽  
Author(s):  
A D Martin ◽  
E H Allan ◽  
M A Titheradge
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Active Form ◽  
Specific Inhibitor ◽  
Hormone Treatment ◽  
Dexamethasone Treatment ◽  
Pyruvate Metabolism ◽  
Glucocorticoid Treatment ◽  
Pyruvate Carboxylation ◽  
Liver Homogenates ◽  
Stimulation Of

Treatment of rats for 3 h with dexamethasone was shown to stimulate both pyruvate carboxylation and decarboxylation in the subsequently isolated mitochondria. The effect of hormone treatment on pyruvate carboxylation was also apparent in liver homogenates assayed within minutes of killing the animal and was independent of the temperature at which the assay was performed, suggesting that it was not an artifact of the mitochondrial preparation procedure. The stimulation of both aspects of pyruvate metabolism in the intact organelle was independent of the induction of either pyruvate carboxylase or pyruvate dehydrogenase. Similarly, there was no change in the percentage of pyruvate dehydrogenase in the active form, indicating that the effect of steroid treatment on pyruvate oxidation was not via changes in the degree of phosphorylation of the enzyme. Adrenalectomizing the animals for a period of 14 days before the experiment had no effect on either parameter. Glucocorticoid treatment of the animals increased the rate of pyruvate uptake into the mitochondria, as measured by the titration of pyruvate metabolism with alpha-cyano-4-hydroxycinnamate, a specific inhibitor of the pyruvate translocator. It also increased the intramitochondrial concentrations of acetyl-CoA and ATP and led to an elevated [ATP]/[ADP] ratio within the mitochondria. It is suggested that both enzymes of pyruvate metabolism exist in the mitochondria under considerable restraint and that glucocorticoids act to relieve this restraint by alterations in substrate supply and the intramitochondrial concentrations of effector molecules.

scholarly journals The intramitochondrial volume measured using sucrose as an extramitochondrial marker overestimates the true matrix volume determined with mannitol

1983 ◽  
Vol 214 (2) ◽  
pp. 387-393 ◽  
Author(s):  
A P Halestrap ◽  
P T Quinlan
Keyword(s):  
Liver Mitochondria ◽  
The Other ◽  
Heart Mitochondria ◽  
Protonmotive Force ◽  
Mitochondrial Matrix ◽  
Matrix Volume ◽  
The Matrix ◽  
The Mean ◽  
Isolated Liver

The matrix volume of isolated liver and heart mitochondria has been estimated at various osmolarities and in various osmotic supports using 36Cl- and [14C]sucrose, D-mannitol, D-3-methoxyglucose and choline as extramitochondrial markers. The use of 3-methoxyglucose was only possible at 0 degree C since it entered mitochondria at physiological temperatures. All extramitochondrial markers used gave linear plots of apparent matrix volume against the reciprocal of the osmolarity, but the slope of this plot was greater when sucrose was used than with the other extramitochondrial markers. When extrapolated to infinite osmolarity the mean matrix volume was zero when mannitol was used, but about 0.6 microliter/mg of protein for sucrose and Cl- and -0.4 microliter/mg of protein when choline was used. At physiological osmolarity (about 330 m-osmol) the mean matrix volume of de-energized liver mitochondria in KCl medium estimated using mannitol was 0.46 microliter/mg of protein, whereas that obtained using sucrose was 1.68 microliters/mg of protein. Values in mannitol, choline and sucrose media were similar when mannitol but not sucrose was used as extramitochondrial marker. It is argued that the 3H2O/[14C]mannitol space more accurately reflects the true mitochondrial matrix volume than does the 3H2O/[14C]sucrose space. The consequences of this for measurements of the protonmotive force and the intramitochondrial concentration of metabolites are discussed.

scholarly journals Intramitochondrial regulation of fatty acid β-oxidation occurs between flavoprotein and ubiquinone A role for changes in the matrix volume

1986 ◽  
Vol 239 (3) ◽  
pp. 559-565 ◽  
Author(s):  
A P Halestrap ◽  
J L Dunlop
Keyword(s):  
Fatty Acid ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Hormonal Stimulation ◽  
Terminal Electron Acceptor ◽  
Matrix Volume ◽  
The Matrix ◽  
Range Of Values ◽  
Stimulation Of

Rat liver mitochondria were incubated in media of different osmolarities and in the presence of various substrates. Rates of oxygen consumption and mitochondrial matrix volumes were measured in the presence and absence of ADP and uncoupler. Duroquinol oxidation was insensitive to matrix volume, whereas other substrates tested showed increased rates of oxidation when the matrix volume increased from 1.0 to 1.5 microliter/mg of protein; this is the range of values measured in situ [Quinlan, Thomas, Armston & Halestrap (1983) Biochem. J. 214, 395-404]. Palmitoylcarnitine, octanoate and butyrate oxidations were particularly sensitive to the matrix volume, increasing from negligible rates to maximal rates within this range. Swelling induced by K+ uptake also stimulated palmitoylcarnitine oxidation. A similar effect of volume on substrate oxidation was seen when ferricyanide in the presence or absence of ubiquinone-1 replaced oxygen as terminal electron acceptor. Measurement of flavoprotein reduction (A 460-480) demonstrated that the locus of the effect of matrix volume is between the electron-transfer flavoprotein and ubiquinone. It is suggested that volume-mediated regulation of fatty acid and proline oxidation may be an important component of the hormonal stimulation of their oxidation.

scholarly journals Regulation of glycine catabolism in rat liver mitochondria

1992 ◽  
Vol 283 (2) ◽  
pp. 435-439 ◽  
Author(s):  
M Jois ◽  
H S Ewart ◽  
J T Brosnan
Keyword(s):  
Rat Liver ◽  
Fold Increase ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Mitochondrial Matrix ◽  
Isolated Rat Liver ◽  
Matrix Volume ◽  
Isolated Rat ◽  
Stimulation Of

1. The catabolism of glycine was studied in isolated rat liver mitochondria by measuring release of 14CO2 from [1-14C]-glycine. Incubation of mitochondria in a medium containing 0.5 microM free Ca2+ resulted in an 8-fold increase in the rate of degradation of glycine. Intraperitoneal injection of glucagon (33 or 100 micrograms/100 g body wt.) 25 min before killing of rats also resulted in a 3-fold or 10-fold (depending on dosage) increase in the rate of catabolism of glycine. 2. Both the stimulation by free Ca2+ and that by injection of glucagon in vivo were dependent on phosphate in the incubation medium. This requirement for phosphate was specific, as replacement of phosphate by other permeant anions such as thiocyanate and acetate did not permit the stimulation. The phosphate-dependent stimulation of glycine catabolism by Ca2+ was also evident when mitochondria were incubated in the absence of K+. 3. Mitochondria isolated from rats previously injected with glucagon showed elevated rates of degradation of glycine even in the presence of rotenone, provided that regeneration of NAD+ was affected by providing acetoacetate. 4. Hypo-osmolarity of the medium markedly stimulated the rate of degradation of glycine by mitochondria. Although hypo-osmolarity-induced stimulation of glycine degradation was accompanied by parallel changes in mitochondrial matrix volume, no measurable changes in matrix volume were observed in mitochondria stimulated either by free Ca2+ (0.5 microM) or by injection of glucagon in vivo. Furthermore, Ca2+ stimulated glycine decarboxylation in mitochondria exposed to either hyper-osmolar (410 mosmol) or hypo-osmolar (210 mosmol) conditions. Although hyper-osmolarity decreased and hypo-osmolarity increased matrix volume, stimulation of glycine degradation by Ca2+ was not associated with any further changes in matrix volume. 5. These data demonstrate that the regulation of hepatic glycine oxidation by glucagon and by free Ca2+ is largely independent of changes in mitochondrial matrix volume.

scholarly journals Stimulation of pyruvate transport in metabolizing mitochondria through changes in the transmembrane pH gradient induced by glucagon treatment of rats

1978 ◽  
Vol 172 (3) ◽  
pp. 389-398 ◽  
Author(s):  
A P Halestrap
Keyword(s):  
Membrane Potential ◽  
Respiratory Chain ◽  
Maximum Rate ◽  
Liver Mitochondria ◽  
Ph Gradient ◽  
O2 Uptake ◽  
Pyruvate Metabolism ◽  
The Matrix ◽  
Stimulation Of

Glucagon treatment of rats allowed the isolation of liver mitochondria with enhanced rates of pyruvate metabolism measured in either sucrose or KCl media. No change in the activity of the pyruvate carrier itself was apparent, but under metabolizing conditions, use of the inhibitor of pyruvate transport, alpha-cyano-4-hydroxycinnamate, demonstrated that pyruvate transport limited the rate of pyruvate metabolism. The maximum rate of transport under metabolizing conditions was enhanced by glucagon treatment. Problems involved in measuring the transmembrane pH gradient under metabolizing conditions are discussed and a variety of techniques are used to estimate the matrix pH. From the distribution of methylamine, ammonia and D-lactate and the Ki for inhibition by alpha-cyano-4-hydroxycinnamate it is concluded that the matrix is more acid than the medium and that the pH of the matrix rises after glucagon treatment. The increase in matrix pH stimulates pyruvate transport. The membrane potential, ATP concentration and O2 uptake were also increased under metabolizing conditions in glucagon-treated mitochondria. These changes were correlated with a stimulation of the respiratory chain which can be observed in uncoupled mitochondria [Yamazaki (1975) J. Biol. Chem. 250, 7924–7930]. The mitochondrial Mg2+ content (mean +/- S.E.M.) was increased from 38.8 +/- 1.2 (n = 26) to 47.5 +/- 2.0 (n = 26) ng-atoms/mg by glucagon and the K+ content from 126.7 +/- 10.3 (n = 19) ng-atoms/mg. This may represent a change in membrane potential induced by glucagon in vivo. The physiological significance of these results in the control of gluconeogenesis is discussed.

scholarly journals Effect of glucagon on metabolite compartmentation in isolated rat liver cells during gluconeogenesis from lactate

1977 ◽  
Vol 166 (2) ◽  
pp. 225-235 ◽  
Author(s):  
Elmar A. Siess ◽  
Dietrich G. Brocks ◽  
Herbert K. Lattke ◽  
Otto H. Wieland
Keyword(s):  
Adenine Nucleotides ◽  
Isolated Hepatocytes ◽  
Cell Fractionation ◽  
Acetyl Coa ◽  
Isolated Rat Liver ◽  
Rat Liver Cells ◽  
The Matrix ◽  
Isolated Rat ◽  
Reduced State ◽  
Stimulation Of

1. The subcellular distribution of adenine nucleotides, acetyl-CoA, CoA, glutamate, 2-oxoglutarate, malate, oxaloacetate, pyruvate, phosphoenolpyruvate, 3-phosphoglycerate, glucose 6-phosphate, aspartate and citrate was studied in isolated hepatocytes in the absence and presence of glucagon by using a modified digitonin procedure for cell fractionation. 2. In the absence of glucagon, the cytosol contains about two-thirds of cellular ATP, some 40–50% of ADP, acetyl-CoA, citrate and phosphoenolpyruvate, more than 75% of total 2-oxoglutarate, glutamate, malate, oxaloacetate, pyruvate, 3-phosphoglycerate and aspartate, and all of glucose 6-phosphate. 3. In the presence of glucagon the cytosolic space shows an increase in the content of malate, phosphoenolpyruvate and 3-phosphoglycerate by more than 60%, and those of aspartate and glucose 6-phosphate rise by about 25%. Other metabolites remain unchanged. After glucagon treatment, cytosolic pyruvate is decreased by 37%, whereas glutamate and 2-oxoglutarate decrease by 70%. The [NAD+]/[NADH] ratios calculated from the cytosolic concentrations of the reactants of lactate dehydrogenase and malate dehydrogenase were the same. Glucagon shifts this ratio and also that of the [NADP+]/[NADPH] couple towards a more reduced state. 4. In the mitochondrial space glucagon causes an increase in the acetyl-CoA and ATP contents by 25%, and an increase in [phosphoenolpyruvate] by 50%. Other metabolites are not changed by glucagon. Oxaloacetate in the matrix is only slightly decreased after glucagon, yet glutamate and 2-oxoglutarate fall to about 25% of the respective control values. The [NAD+]/[NADH] ratios as calculated from the [3-hydroxybutyrate]/[acetoacetate] ratio and from the matrix [malate]/[oxaloacetate] couple are lowered by glucagon, yet in the latter case the values are about tenfold higher than in the former. 5. Glucagon and oleate stimulate gluconeogenesis from lactate to nearly the same extent. Oleate, however, does not produce the changes in cellular 2-oxoglutarate and glutamate as observed with glucagon. 6. The changes of the subcellular metabolite distribution after glucagon are compatible with the proposal that the stimulation of gluconeogenesis results from as yet unknown action(s) of the hormone at the mitochondrial level in concert with its established effects on proteolysis and lipolysis.

scholarly journals The Sorting of Mitochondrial DNA and Mitochondrial Proteins in Zygotes: Preferential Transmission of Mitochondrial DNA to the Medial Bud

1998 ◽  
Vol 142 (3) ◽  
pp. 613-623 ◽  
Author(s):  
Koji Okamoto ◽  
Philip S. Perlman ◽  
Ronald A. Butow
Keyword(s):  
Mitochondrial Dna ◽  
Fluorescent Protein ◽  
Yeast Cells ◽  
Mitochondrial Matrix ◽  
Outer Membranes ◽  
Marker Proteins ◽  
The Matrix ◽  
Green Fluorescent ◽  
Mitochondrial Reticulum

Green fluorescent protein (GFP) was used to tag proteins of the mitochondrial matrix, inner, and outer membranes to examine their sorting patterns relative to mtDNA in zygotes of synchronously mated yeast cells in ρ+ × ρ0 crosses. When transiently expressed in one of the haploid parents, each of the marker proteins distributes throughout the fused mitochondrial reticulum of the zygote before equilibration of mtDNA, although the membrane markers equilibrate slower than the matrix marker. A GFP-tagged form of Abf2p, a mtDNA binding protein required for faithful transmission of ρ+ mtDNA in vegetatively growing cells, colocalizes with mtDNA in situ. In zygotes of a ρ+ × ρ+ cross, in which there is little mixing of parental mtDNAs, Abf2p–GFP prelabeled in one parent rapidly equilibrates to most or all of the mtDNA, showing that the mtDNA compartment is accessible to exchange of proteins. In ρ+ × ρ0 crosses, mtDNA is preferentially transmitted to the medial diploid bud, whereas mitochondrial GFP marker proteins distribute throughout the zygote and the bud. In zygotes lacking Abf2p, mtDNA sorting is delayed and preferential sorting is reduced. These findings argue for the existence of a segregation apparatus that directs mtDNA to the emerging bud.

scholarly journals Primary structure requirements for correct sorting of the yeast mitochondrial protein ADH III to the yeast mitochondrial matrix space.

1987 ◽  
Vol 7 (1) ◽  
pp. 294-304 ◽  
Author(s):  
D Pilgrim ◽  
E T Young
Keyword(s):  
Amino Acids ◽  
Enzyme Activity ◽  
Amino Acid ◽  
Proteolytic Processing ◽  
Mitochondrial Matrix ◽  
Wild Type ◽  
Mature Form ◽  
Amino Terminal ◽  
The Matrix

Alcohol dehydrogenase isoenzyme III (ADH III) in Saccharomyces cerevisiae, the product of the ADH3 gene, is located in the mitochondrial matrix. The ADH III protein was synthesized as a larger precursor in vitro when the gene was transcribed with the SP6 promoter and translated with a reticulocyte lysate. A precursor of the same size was detected when radioactively pulse-labeled proteins were immunoprecipitated with anti-ADH antibody. This precursor was rapidly processed to the mature form in vivo with a half-time of less than 3 min. The processing was blocked if the mitochondria were uncoupled with carbonyl cyanide m-chlorophenylhydrazone. Mutant enzymes in which only the amino-terminal 14 or 16 amino acids of the presequence were retained were correctly targeted and imported into the matrix. A mutant enzyme that was missing the amino-terminal 17 amino acids of the presequence produced an active enzyme, but the majority of the enzyme activity remained in the cytoplasmic compartment on cellular fractionation. Random amino acid changes were produced in the wild-type presequence by bisulfite mutagenesis of the ADH3 gene. The resulting ADH III protein was targeted to the mitochondria and imported into the matrix in all of the mutants tested, as judged by enzyme activity. Mutants containing amino acid changes in the carboxyl-proximal half of the ADH3 presequence were imported and processed to the mature form at a slower rate than the wild type, as judged by pulse-chase studies in vivo. The unprocessed precursor appeared to be unstable in vivo. It was concluded that only a small portion of the presequence contains the necessary information for correct targeting and import. Furthermore, the information for correct proteolytic processing of the presequence appears to be distinct from the targeting information and may involve secondary structure information in the presequence.

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