scholarly journals The mechanism of the hormonal activation of respiration in isolated hepatocytes and its importance in the regulation of gluconeogenesis

1986 ◽  
Vol 236 (3) ◽  
pp. 789-800 ◽  
Author(s):  
P T Quinlan ◽  
A P Halestrap
Keyword(s):  
Cytochrome C ◽  
Electron Flow ◽  
Isolated Hepatocytes ◽  
Liver Mitochondria ◽  
Acid Oxidation ◽  
O2 Consumption ◽  
Cytochrome Bc1 Complex ◽  
Fluorescence Response ◽  
Time Courses ◽  
Stimulation Of

The effects of hormones on the cytochrome spectra of isolated hepatocytes were recorded under conditions of active gluconeogenesis from L-lactate. Glucagon, phenylephrine, vasopressin and valinomycin, at concentrations that caused stimulation of gluconeogenesis, increased the reduction of the components of the cytochrome bc1 complex, just as has been observed in liver mitochondria isolated from glucagon-treated rats [Halestrap (1982) Biochem. J. 204, 37-47]. The effects of glucagon and phenylephrine were additive. The time courses of the increased reduction of cytochrome c/c1 and NAD(P)H/NAD(P)+ caused by hormones, valinomycin, A23187 and ethanol were measured by dual-beam spectrophotometry and fluorescence respectively. Ethanol (14 mM) produced a substantial rise in NAD(P)H fluorescence, beta-hydroxybutyrate/acetoacetate and lactate/pyruvate ratios, no change in cytochrome c/c1 reduction, a 10% decrease in O2 consumption and a 60% decrease in gluconeogenesis. Glucagon, phenylephrine and vasopressin caused a substantial and transient rise in NAD(P)H fluorescence, but a sustained increase in cytochrome c/c1 reduction and the rates of O2 consumption and gluconeogenesis. The transience of the fluorescence response was greater in the absence of Ca2+, when the cytochrome c/c1 response also became transient. The fluorescence response was smaller and less transient, but the cytochrome c/c1 response was greater, in the presence of fatty acids. Both responses were greatly decreased by the presence of 1 mM-pent-4-enoate. Valinomycin (2.5 nM) caused a decrease in NAD(P)H fluorescence coincident with an increase in cytochrome c/c1 reduction and the rate of gluconeogenesis and O2 consumption. A23187 (7.5 mM) caused increases in both NAD(P)H fluorescence and cytochrome c/c1 reduction. The effects of hormones and valinomycin on the time courses of NAD(P)H fluorescence, cytochrome c/c1 reduction and light-scattering by hepatocytes were compared with those of 0.5 microM-Ca2+ or 1 nM-valinomycin on the same parameters of isolated liver mitochondria. It is concluded that hormones increase respiration by hepatocytes in a biphasic manner. An initial Ca2+-dependent activation of mitochondrial dehydrogenases rapidly increases the mitochondrial [NADH], which is followed by a volume-mediated stimulation of fatty acid oxidation and electron flow between NADH and cytochrome c. 10. Amytal (0.5 mM) was able to reverse the effects of hormones on the reduction of cytochromes c/c1 and the rates of gluconeogenesis and O2 consumption without significantly lowering tissue [ATP].(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Steady-state H+/O stoichiometry of liver mitochondria

1981 ◽  
Vol 200 (3) ◽  
pp. 539-546 ◽  
Author(s):  
M K Al-Shawi ◽  
M D Brand
Keyword(s):  
Steady State ◽  
Cytochrome C ◽  
Consumption Rate ◽  
Initial Rate ◽  
Electron Flow ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
O2 Consumption ◽  
Ejection Rate ◽  
Novel Method

We have measured the H+/O stoichiometry of rat liver mitochondria respiring in a steady-state, using a novel method. This involves measuring the initial rate of H+ back-flow into mitochondria after respiratory inhibition, with the assumption that this is equal to the steady-state H+-ejection rate. Division by the steady-state O2-consumption rate yields the H+/O ratio. The H+/O values obtained were: 8.3 +/- 1.0 (mean +/- S.E.M.) for 3-hydroxybutyrate: 8.2 +/- 0.7 for glutamate plus malate; 6.0 +/- 0.2 for succinate; 4.1 +/- 0.3 for ascorbate/tetramethylphenylenediamine and 3.0 +/- 0.1 for ascorbate/ferrocyanide. These values correspond to H+/O stoichiometries for electron flow to oxygen from NAD+-linked substrates, succinate and cytochrome c of 8, 6 and 2 (charge/O ratio = 4) respectively.

Reversal of mitochondrial swelling associated with fatty acid oxidation. II. Effects of cytochrome c and carnitine on contraction of fatty acid swollen mitochondria

1968 ◽  
Vol 46 (9) ◽  
pp. 1151-1160 ◽  
Author(s):  
Misako Nakatani ◽  
W. C. McMurray
Keyword(s):  
Fatty Acid ◽  
Cytochrome C ◽  
Energy Requirement ◽  
Liver Mitochondria ◽  
High Energy ◽  
Fatty Acyl ◽  
Water Soluble ◽  
Rat Liver Mitochondria ◽  
Acid Oxidation ◽  
Swelling Agent

Rat liver mitochondria undergo reversible swelling in the presence of a fatty acyl CoA generating system. Contraction of the swollen mitochondria was observed on the addition of either carnitine or cytochrome c. At low concentrations the two agents acted synergistically. At high concentrations cytochrome c completely replaced the requirement for carnitine.Cytochrome c also promoted the contraction of mitochondria swollen in the presence of fatty acid alone, provided that either ATP or ADP was added to initiate the contraction. The stimulation by cytochrome c was greater in the presence of ADP, and the contraction was more sensitive to respiratory inhibitors or dinitrophenol but was less sensitive to oligomycin than in the presence of ATP. Studies of the metabolism of 14C-labelled palmitate during cytochrome c induced contraction showed that decreases in mitochondrial-bound fatty acid and corresponding increases in water-soluble metabolites coincided with the reversal of swelling. The results indicated that the energy requirement for mitochondrial contraction in the presence of cytochrome c was provided by generation of high-energy intermediates coupled to oxidation of the fatty acid swelling agent.

scholarly journals The regulation of the oxidation of fatty acids and other substrates in rat heart mitochondria by changes in the matrix volume induced by osmotic strength, valinomycin and Ca2+

1987 ◽  
Vol 244 (1) ◽  
pp. 159-164 ◽  
Author(s):  
A P Halestrap
Keyword(s):  
Rat Heart ◽  
Electron Flow ◽  
Liver Mitochondria ◽  
Heart Mitochondria ◽  
Acid Oxidation ◽  
Maximal Effect ◽  
Physiological Conditions ◽  
Matrix Volume ◽  
The Matrix ◽  
Rat Heart Mitochondria

1. The rate of ADP-stimulated respiration with various substrates and the matrix volume of rat heart mitochondria were measured over a range of osmolarities of the medium. 2. The rate of oxidation of palmitoylcarnitine (in the presence of malate) was stimulated 7-fold by increasing the matrix volume from 0.6 to 1.0 microliter/mg of protein. Oxidation of octanoate showed a similar sensitivity to the matrix volume, whereas oxidation of other substrates showed little sensitivity until the volume fell below 0.7 microliter/mg of protein. 3. The matrix volume of heart mitochondria incubated under physiological conditions was about 0.8 microliter/mg of protein. 4. Low concentrations of valinomycin added to mitochondria incubated under such physiological conditions could activate the rate of ADP-stimulated palmitoylcarnitine oxidation by at least 100%. 5. Decreasing the matrix volume increased the reduction of the electron-transferring flavoprotein (ETF), suggesting an effect on electron flow between ETF and ubiquinone, as has been observed for liver mitochondria [Halestrap & Dunlop (1986) Biochem. J. 239, 559-565]. 6. A rapid decrease in light-scattering by heart mitochondria incubated in State 4 was induced by addition of Ca2+, reaching 50% of the maximal effect after about 30 s at 30 degrees C and with K0.5 for Ca2+ of 0.3 microM. This was not associated with a change in matrix volume, and is discussed in terms of a conformational change whose identity remains to be determined. 7. However, incubation of heart mitochondria at 37 degrees C in the presence of 0.65 microM-Ca2+ for 4 min did increase the matrix volume significantly, by 0.181 +/- 0.029 microliter/mg of protein (n = 7, P less than 0.001), similar to the Ca2+-induced changes observed with liver mitochondria [Halestrap, Quinlan, Whipps & Armston (1986) Biochem. J. 236, 779-787]. 8. The possible significance of these results in the co-ordinate regulation of fatty acid oxidation and the citric acid cycle in the heart responding to increased work load or hormonal stimulation is discussed.

scholarly journals The nature of the stimulation of the respiratory chain of rat liver mitochondria by glucagon pretreatment of animals

1982 ◽  
Vol 204 (1) ◽  
pp. 37-47 ◽  
Author(s):  
A P Halestrap
Keyword(s):  
Respiratory Chain ◽  
Electron Flow ◽  
Liver Mitochondria ◽  
Complex Iii ◽  
Secondary Site ◽  
Rat Liver Mitochondria ◽  
Succinate Oxidation ◽  
Q Cycle ◽  
Cytochromes C ◽  
Stimulation Of

1. Studies on the cytochrome spectra of liver mitochondria from control and glucagon-treated rats in State 4, State 3 and in the presence of uncoupler are reported. 2. The stimulation of electron flow between cytochromes c1 and c observed previously [Halestrap (1978) Biochem. J. 172, 399-405] was shown to be an artefact of Ca2+-induced swelling of mitochondria. 3. When precautions were taken to prevent such swelling, glucagon treatment was shown to enhance the reduction of cytochromes c, c1 and b558 in both State 3 and uncoupled conditions with either succinate or glutamate + malate as substrate. An increase in the reduction of cytochromes b562 and b566 was also seen in some, but not all, experiments. 4. In State 4 with succinate but not glutamate + malate as substrate, cytochromes c, c1, b558, b562 and b566 showed increased reduction. 5. Glucagon stimulated oxidation of duroquinol and palmitoylcarnitine by intact mitochondria and of NADH by disrupted mitochondria. 6. No effect of glucagon on succinate dehydrogenase activity or the temperature-dependence of succinate oxidation could be detected. 7. Glucagon enhanced the inhibition of the respiratory chain by colletotrichin, but not antimycin or 8-heptyl-4-hydroxyquinoline N-oxide. 8. These results are interpreted in terms of a primary stimulation by glucagon of the ‘Q cycle’ [Mitchell (1976) J. Theor. Biol. 62, 827-367] within Complex III (ubiquinol:cytochrome c oxidoreductase) and a secondary site of action involving stimulation of electron flow into Complex III from the ubiquinone pool. 9. Ageing of mitochondria, hyperosmotic treatment or addition of 20 mM-benzyl alcohol opposed the effects of glucagon treatment on cytochrome spectra and colletotrichin inhibition of respiration. 10. These results support the hypothesis that glucagon exerts its effects on the mitochondria by perturbing the membrane structure.

scholarly journals The effect of cytochrome c and its ‘dimer’ on electron transfer and energy transformation

1972 ◽  
Vol 126 (3) ◽  
pp. 709-716 ◽  
Author(s):  
T. Shur-Perek ◽  
Y. Avi-Dor
Keyword(s):  
Electron Transfer ◽  
Electron Transport ◽  
Cytochrome C ◽  
Proton Translocation ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Energy Transformation ◽  
Early Step ◽  
Electron Carrier ◽  
Stimulation Of

A preparation that contained cytochrome c, mainly in the form of its ‘dimer’, was studied and compared with native cytochrome c with respect to its ability to support electron transfer and energy transformation in cytochrome c-depleted rat liver mitochondria. When the depleted mitochondria were titrated with either cytochrome c or the ‘dimer’, the extent of coupling between respiration and phosphorylation was enhanced, as manifested by an increase in the P/O ratio. The ‘dimer’ was relatively ineffective as an electron carrier in the respiratory system, but it was as effective as cytochrome c in reconstitution of oxidative phosphorylation in depleted mitochondria. Addition of ‘dimer’ to the depleted mitochondria, in the presence of a low, non-saturating concentration of cytochrome c, increased the P/O ratio without concomitant stimulation of respiration. Both cytochrome c and the ‘dimer’ stimulated spontaneous swelling and electron transport-driven proton translocation in depleted mitochondria. The pattern of action of cytochrome c and its ‘dimer’ is in accord with the assumption that they affect an early step in energy conservation.

scholarly journals The H+/e− stoicheiometry of respiration-linked proton translocation in the cytochrome system of mitochondria

1980 ◽  
Vol 192 (1) ◽  
pp. 203-218 ◽  
Author(s):  
Sergio Papa ◽  
Ferruccio Guerrieri ◽  
Michele Lorusso ◽  
Gianfranco Izzo ◽  
Domenico Boffoli ◽  
...  
Keyword(s):  
Cytochrome C ◽  
Aqueous Phase ◽  
Electron Flow ◽  
Proton Translocation ◽  
O2 Consumption ◽  
Proton Release ◽  
Succinate Oxidation ◽  
Neutral Ph ◽  
Ferricytochrome C ◽  
The Matrix

1. The →H+/e− quotients for proton release from mitochondria associated with electron flow from succinate and duroquinol to O2, ferricyanide or ferricytochrome c, and from NNN′N′-tetramethyl-p-phenylenediamine+ascorbate to O2, were determined from rate measurements of electron flow and proton translocation. 2. Care was taken to avoid, or to take into account, unrelated electron flow and proton translocation, which might take place in addition to the oxido-reductions that were the subject of our analysis. Spectrophotometric techniques were chosen to provide accurate measurement of the rate of consumption of oxidants and reductants. The rate of proton translocation was measured with fast pH meters with a precision of 10−3 pH unit. 3. The →H+/O quotient for succinate or duroquinol oxidation was, at neutral pH, 4, when computed on the basis of spectrophotometric determinations of the rate of O2 consumption or duroquinol oxidation. Higher →H+/O quotients for succinate oxidation, obtained from polarographic measurements of O2 consumption, resulted from underestimation of the respiratory rate. 4. The →H+/2e− quotient for electron flow from succinate and duroquinol to ferricyanide or ferricytochrome c ranged from 3.9 to 3.6. 5. Respiration elicited by NNN′N′-tetramethyl-p-phenylenediamine+ascorbate by antimycin-inhibited mitochondria resulted in extra proton release in addition to that produced for oxidation of ascorbate to dehydroascorbate. Accurate spectrophotometric measurement of respiration showed that the →H+/e− ratio was only 0.25 and not 0.7–1.0 as obtained with the inadequate polarographic assay of respiration. Proton release was practically suppressed when mitochondria were preincubated aerobically in the absence of antimycin. Furthermore, the rate of scalar proton consumption for water production was lower than that expected from the stoicheiometry. Thus the extra proton release observed during respiration elicited by NNN′N′-tetramethyl-p-phenylenediamine+ascorbate is caused by oxidation of endogenous hydrogenated reductants. 6. It is concluded that (i) the →H+/O quotient for the cytochrome system is, at neutral pH, 4 and not 6 or 8 as reported by others; (ii) all the four protons are released during electron flow from quinol to cytochrome c; (iii) the oxidase transfers electrons from cytochrome c to protons from the matrix aqueous phase and does not pump protons from the matrix to the outer aqueous phase.

Regulation of fatty acid oxidation in isolated hepatocytes and liver mitochondria from newborn rabbits

1987 ◽  
Vol 165 (1) ◽  
pp. 201-207 ◽  
Author(s):  
Catherine HERBIN ◽  
Jean-Paul PEGORIER ◽  
Pierre-Henri DUEE ◽  
Claude KOHL ◽  
Jean GIRARD
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Oxidation ◽  
Isolated Hepatocytes ◽  
Liver Mitochondria ◽  
Acid Oxidation

Flux through glycine cleavage system in isolated hepatocytes: effects of glucagon, cAMP, and calcium

1990 ◽  
Vol 68 (2) ◽  
pp. 543-546 ◽  
Author(s):  
Markandeya Jois ◽  
Beatrice Hall ◽  
Vaughn M. Collett ◽  
John T. Brosnan
Keyword(s):  
Acid Metabolism ◽  
Rat Hepatocytes ◽  
Isolated Hepatocytes ◽  
Liver Mitochondria ◽  
Glycine Cleavage System ◽  
Isolated Rat Hepatocytes ◽  
Maximal Stimulation ◽  
Glycine Cleavage ◽  
Camp Levels ◽  
Stimulation Of

The hepatic glycine cleavage system (GCS) is the principal route for the metabolism of glycine in mammals. Flux through the GCS in isolated rat hepatocytes was stimulated by about 100% by glucagon (10−7 M), forskolin (10−4 M), and dibutyryl cAMP (10−4 M). The stimulation of flux through the GCS by these agents was accompanied by marked elevation of cellular cAMP levels. A significant correlation was observed between increased cellular cAMP levels induced by glucagon and stimulation of flux through the GCS by glucagon. Exclusion of calcium from the incubation medium reduced the basal flux by 38%, but did not affect the degree of stimulation of flux through the GCS by glucagon. A single intraperitoneal injection of glucagon to rats prior to isolation of hepatocytes resulted in a 76% stimulation of flux through the GCS. These hepatocytes with stimulated flux through the GCS showed reduced sensitivity for further stimulation by glucagon. Half-maximal stimulation of flux through the GCS occurred at 3.8 ± 1.1 and 8.5 ± 1.4 nM glucagon in hepatocytes isolated from control and glucagon-injected rats, respectively. We conclude that cAMP is involved in the regulation of flux through the GCS by gluagon.Key words: amino acid, metabolism, liver, mitochondria, hormones.

scholarly journals The role of intermediates in mitochondrial fatty acid oxidation

1975 ◽  
Vol 150 (1) ◽  
pp. 77-88 ◽  
Author(s):  
K K Stanley ◽  
P K Tubbs
Keyword(s):  
Fatty Acid ◽  
Short Pulse ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Acid Oxidation ◽  
Mitochondrial Fatty Acid Oxidation ◽  
Mitochondrial Fatty Acid ◽  
Time Courses ◽  
Long Chain

1. Rat liver mitochondria oxidizing [16-14C]palmitoylcarnitine accumulate saturated long-chain thiester intermediates which may be detected by radio-g.1.c.2. Time-courses of intermediate accumulation display no product-precursor relationships and the end product, measured as [14C]citrate, is produced without a detectable initial lag. 3. A short pulse of [16-14C]palmitoylcarnitine followed by unlabelled palmitoylcarnitine showed that the observed intermediates(at least in the greater part)were not the direct precursors of [14C]citrate. 4. The quantity of saturated intermediates depended on the total accumulated flux of acyl units through the pathway provided that some mitochondrial CoA and unused substrate remained. 5. In the presence of rotenone and carnitine, 2-unsaturated, 3-unsaturated and 3-hydroxy intermediates were formed as well as saturated intermediates…

Effects of 5-5'-Diphenyl-2-thiohydantoin (DPTH) and Thyroxine on the Ultrastructure and Chemical Composition of Rat Liver

1971 ◽  
Vol 29 ◽  
pp. 570-571
Author(s):  
E. A. Elfont ◽  
R. B. Tobin ◽  
D. G. Colton ◽  
M. A. Mehlman
Keyword(s):  
Chemical Composition ◽  
Rat Liver ◽  
Future Study ◽  
Mode Of Action ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Effective Inhibitor ◽  
Biochemical Effects ◽  
Glycerophosphate Dehydrogenase ◽  
Stimulation Of

Summary5,-5'-diphenyl-2-thiohydantoin (DPTH) is an effective inhibitor of thyroxine (T4) stimulation of α-glycerophosphate dehydrogenase in rat liver mitochondria. Because this finding indicated a possible tool for future study of the mode of action of thyroxine, the ultrastructural and biochemical effects of DPTH and/or thyroxine on rat liver mere investigated.Rats were fed either standard or DPTH (0.06%) diet for 30 days before T4 (250 ug/kg/day) was injected. Injection of T4 occurred daily for 10 days prior to sacrifice. After removal of the liver and kidneys, part of the tissue was frozen at -50°C for later biocheailcal analyses, while the rest was prefixed in buffered 3.5X glutaraldehyde (390 mOs) and post-fixed in buffered 1Z OsO4 (376 mOs). Tissues were embedded in Araldlte 502 and the sections examined in a Zeiss EM 9S.Hepatocytes from hyperthyroid rats (Fig. 2) demonstrated enlarged and more numerous mitochondria than those of controls (Fig. 1). Glycogen was almost totally absent from the cytoplasm of the T4-treated rats.

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