Rapid thyroid-hormone effect on mitochondrial and cytosolic ATP/ADP ratios in the intact liver cell.

The effect of thyroid-hormone application on cytosolic and mitochondrial ATP/ADP ratio was investigated in rat liver in vivo and in the isolated perfused organ. In vivo the ATP/ADP ratio in livers from hypothyroid rats was 0.84 +/- 0.08 in the mitochondrial matrix and 5.6 +/- 0.9 in the cytosol, as was observed in euthyroid controls. In contrast, hyperthyroidism was followed by a significant decrease in the mitochondrial and by an increase in the cytosolic ATP/ADP ratio (to 0.34 +/- 0.06 and 11.3 +/- 2.8 respectively). In the perfused liver from hypothyroid animals, addition of L-3,3',5-tri-iodothyronine in the perfusate also provoked, within 2 h, a significant decrease in the mitochondrial ATP/ADP ratio, whereas the cytosolic ratio was unaffected. From these and previous data in the isolated perfused liver and in isolated mitochondria from hypothyroid and tri-iodothyronine-treated rats it is concluded that thyroid hormones increase mitochondrial respiration and ATP regeneration, which is associated with an acceleration of mitochondrial adenine nucleotide transport and significant alterations in the mitochondrial and cytosolic ATP/ADP ratios.     

Phosphorylation state of cytosolic and mitochondrial adenine nucleotides and of pyruvate dehydrogenase in isolated rat liver cells.

1. Cytosolic and mitochondrial ATP and ADP concentrations of liver cells isolated from normal fed, starved and diabetic rats were determined. 2. The cytosolic ATP/ADP ratio was 6,9 and 10 in normal fed, starved and diabetic rats respectively. 3. The mitochondrial ATP/ADP ratio was 2 in normal and diabetic rats and 1.6 in starved rats. 4. Adenosine increased the cytosolic and lowered the mitochondrial ATP/ADP ratio, whereas atractyloside had the opposite effect. 5. Incubation of the hepatocytes with fructose, glycerol or sorbitol led to a fall in the ATP/ADP ratio in both the cytosolic and the mitochondrial compartment. 6. The interrelationship between the mitochondrial ATP/ADP ratio and the phosphorylation state of pyruvate dehydrogenase in intact cells was studied. 7. In hepatocytes isolated from fed rats an inverse correlation between the mitochondrial ATP/ADP ratio and the active form of pyruvate dehydrogenase (pyruvate dehydrogenase a) was demonstrable on loading with fructose, glycerol or sorbitol. 8. No such correlation was obtained with pyruvate or dihydroxyacetone. For pyruvate, this can be explained by inhibition of pyruvate dehydrogenase kinase. 9. Liver cells isolated from fed animals displayed pyruvate dehydrogenase a activity twice that found in vivo. Physiological values were obtained when the hepatocytes were incubated with albumin-oleate, which also yielded the highest mitochondrial ATP/ADP ratio.     

Mitochondrial and cytosolic ATP/ADP ratios in rat liver in vivo.

The ratio of ATP content/ADP content in livers from unanaesthetized fed rat was 0.9 in the mitochondrial matrix and 6.9 in the cytosol; the values for starved (48 h) animals were 1.0 and 5.9 respectively. The mitochondrial ratios observed in unanaesthetized animals were higher than in haemoglobin-free-perfused liver and lower than in isolated hepatocytes. Possible reasons for these differences may be related to oxygen supply and/or other factors. Further, data from anaesthetized rats with the liver exposed are given: mitochondrial ATP/ADP ratios were decreased with pentobarbital, but less so with ketamine as narcotic agent.     

Gradients of O2 concentration in hepatocytes.

1. Cytochrome P/450-dependent mixed function oxidations of hexobarbital, phenyramidol, and alprenolol in intact hepatocytes were examined at different steady state oxygen concentrations. Apparent Kmo2 values were determined to be 6.4 +/- 1.7, 3.6 +/- 0.6, and 9.8 +/- 1.2 micronM, respectively. 2. Apparent Kmo2 values for metabolism of hexobarbital and alprenolol by liver microsomes were 4.3 +/- 0.4 and 8.7 +/- 0.7 micronM, similar to the corresponding values for whole cells. Therefore, no detectable gradient of O2 concentration exists between extracellular space and endoplasmic reticulum of hepatocytes at these oxygen concentrations. 3. Steady state concentrations of ATP, ADP, AMP, lactate, and pyruvate at different steady state oxygen concentrations were used as indicators of mitochondrial oxygen dependence in intact hepatocytes. Half-maximal changes occurred at [O2] = 12.6 micronM for cytoplasmic [NAD+]/[NADH] (estimated from [lactate]/[pyruvate]), at 7.0 micronM for [ATP]/[ADP], and at 2.8 micronM for adenylate energy charge. The apparent cellular respiratory Kmo2 was 1.90 +/- 0.18 micronM. 4. Comparison of values for oxygen dependence of mitochondrial functions in isolated hepatocytes with published values for isolated mitochondria suggests that a substantial intracellular oxygen gradient exists between the outer cellular membrane and the mitochondrial inner membrane at po2 values below the critical O2 tensions.     

Effect of ATP translocation on citrulline and oxaloacetate synthesis by isolated rat liver mitochondria.

The possibility that the availability of ATP may affect the rate of synthesis of carbamoyl phosphate (measured as citrulline) by carbamoyl phosphate synthase (ammonia) was studied using respiring isolated rat liver mitochondria incubated with added ADP, with hexokinase, glucose, and ATP, or with atractylate, in order to enhance or prevent the efflux of mitochondrial ATP. The effects of these agents were compared with those on oxaloacetate synthesis from pyruvate. Addition of hexokinase, glucose, and ATP to isolated mitochondria resulted in an inhibition of citrulline synthesis which was proportional to the amounts of glucose 6-phosphate formed; under these conditions, matrix ATP and ATP/ADP tended to decrease. The addition of increasing amounts of ADP also resulted in proportional inhibition of citrulline synthesis, but in this case the matrix content of ATP and ADP increased, and ATP/ADP decreased very slightly. In the presence of atractylate, citrulline synthesis was maximal despite a 30% decrease in matrix ATP and ATP/ADP. These effects were observed whether pyruvate, succinate, glutamate, or β-OH-butyrate was used as the respiratory substrate. ADP, the hexokinase system, and atractylate had qualitatively similar but much less pronounced effects on oxaloacetate synthesis from pyruvate. Within the limits of variation observed in these experiments, the rate of synthesis of citrulline appears not to be affected by the matrix content of total ATP, total ADP, or by ATP/ADP. It is affected, however, by the velocity of translocation of ATP into the extramitochondrial medium. These findings suggest that carbamoyl phosphate synthase (ammonia) may be loosely associated with the mitochondrial inner membrane, and may compete for ATP with the ATP-ADP translocator to an extent determined by the extramitochondrial demands for ATP.     

Repartition of ATP, ADP and PO4 in isolated hepatocytes from fed and fasted rats

1. The digitonin fractionation procedure [Zuurendonk, P. F. and Tager, J. M. (1974) Biochim. Biophys. Acta, 333, 393-399] was used to determine the repartition of adenine nucleotides and inorganic phosphate in isolated hepatocytes from fed and fasted rats. 2. This repartition is not significantly modified in the presence of pyruvate or alanine or lactate + pyruvate for isolated hepatocytes from fasted rats. 3. In isolated hepatocytes from fasted rats, the mitochondrial ATP/ADP X PO4 ratio is two-fold lower than in isolated hepatocytes from fed rats. 4. The cytosolic ATP/ADP X PO4 ratio depends on the nutritional state and (or) on the added substrate for neoglucogenesis.     

31P NMR saturation-transfer study of the in situ kinetics of the mitochondrial adenine nucleotide translocase

The exchange of intramitochondrial ATP (ATP(in)) for extramitochondrial ATP (ATP(out)) was measured by using 31P NMR spectroscopy over a range of temperatures in isolated rat liver mitochondria oxidizing glutamate and succinate in the presence of external ATP but no added ADP (state 4). The rate of this exchange is more than an order of magnitude faster than rates reported previously that were determined by using isotopic techniques in the presence of oligomycin, the potent ATPase inhibitor. Differences are ascribed in part to the low levels of matrix ATP present in oligomycin-treated mitochondria. The addition of oligomycin to mitochondrial suspensions decreases intramitochondrial ATP levels from 17 +/- 3 (SEM) nmol/mg of protein in state 4 to 1.51 +/- 0.1 nmol/mg of protein in the presence of inhibitor at 8 degrees C. Simultaneously, transporter flux falls from 960 +/- 55 nmol/min.mg to undetectable levels (less than 300 nmol/min.mg). Although transport rates are much faster when measured by saturation-transfer than by conventional isotopic methods, the enthalpy values obtained by determining the effect of temperature on flux are very similar to those reported in the past that were determined by using isotopic techniques. Intramitochondrial ATP content regulates the rate of the ATP(in)/ATP(out) exchange. At 18 degrees C, the concentration of internal ATP that produces half-maximal transport rate is 6.6 +/- 0.12 nmol/mg of mitochondrial protein. The relationship between substrate concentration and flux is sigmoidal and is 90% saturated at 11.3 +/- 0.18 nmol/mg of mitochondrial protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

ATP-Mg/Pi carrier activity in rat liver mitochondria.

The ATP-Mg/Pi carrier in liver mitochondria is activated by micromolar Ca2+ and mediates net adenine nucleotide transport into and out of the mitochondrial matrix. The purpose of this study was to characterize certain features of ATP-Mg/Pi carrier activity that are essential for understanding how the mitochondrial adenine nucleotide content is regulated. The relative importance of ATP and ADP as transport substrates was investigated using specific trap assays to measure their separate rates of carrier-mediated efflux with Pi as the external counterion. Under energized conditions ATP efflux accounted for 88% of total ATP+ADP efflux. With oligomycin present to lower the matrix ATP/ADP ratio, ATP efflux was eliminated and ADP efflux was relatively unaffected. Mg2+ was stoichiometrically required for ATP influx and is probably transported simultaneously with ATP. Ca2+ and Mn2+ could substitute for the stoichiometric Mg2+ requirement. ADP influx and Pi-induced adenine nucleotide efflux were unaffected by external Mg2+. Experiments with Pi analogues suggested that Pi is transported as the divalent anion, HPO4(2-). The results show that ATP-Mg and divalent Pi are the major transport substrates; the most probable transport mechanism for the ATP-Mg/Pi carrier is an electroneutral exchange. The results are consistent with the hypothesis that the direction and magnitude of net adenine nucleotide movements are determined mainly by the (ATP-Mg)2- and HPO4(2-) concentration gradients across the inner mitochondrial membrane.     

Dependence of mitochondrial and cytosolic adenine nucleotides on oxygen partial pressure in isolated hepatocytes. Application of a new rapid high pressure filtration technique for fractionation.

By using a new rapid high pressure filtration technique, mitochondrial and cytosolic ATP and ADP contents were determined in isolated hepatocytes at different oxygen partial pressures. At 670 mmHg, subcellular adenine nucleotide contents and ATP/ADP ratios were comparable with values obtained with the digitonin fractionation technique. However at lower oxygen partial pressure ADP appears to be rephosphorylated during digitonin fractionation whereas with high pressure filtration fractionation rephosphorylation of ADP is avoided due to shorter fractionation times. Cytosolic and mitochondrial ATP/ADP ratios decrease if oxygen partial pressure is lowered. However the absolute values of ATP/ADP ratios depend critically on the incubation conditions. Thus incubation of hepatocytes in an oxystat system, where oxygen partial pressure is maintained constant by infusing oxygen-saturated medium and the hepatocyte suspension is continuously stirred, yields much higher subcellular and overall ATP/ADP ratios than incubation in Erlenmeyer flasks gassed with different gas mixtures and shaken in a water bath. This is ascribed to limited diffusion of oxygen from the medium into the cell if the suspension is not mixed thoroughly by stirring. The strong dependence of subcellular ATP/ADP ratios on incubation conditions indicates that oxygen may be one rate-controlling factor for oxidative phosphorylation in the intact cell.     

Studies on the effects of coenzyme A-SH: acetyl coenzyme A, nicotinamide adenine dinucleotide: reduced nicotinamide adenine dinucleotide, and adenosine diphosphate: adenosine triphosphate ratios on the interconversion of active and inactive pyruvate dehydrogenase in isolated rat heart mitochondria.

The content of coenzyme A-SH (CoASH) and acetyl-CoA of suspensions of rat heart mitochondria was stabilized by the addition of DL-carnitine and acetyl-DL-carnitine, in the presence of the respiratory inhibitor rotenone. The mitochondrial content of NAD+ and NADH was similarly stabilized by the addition of acetoacetate and DL-3-hydroxybutyrate, and the content of ADP and ATP was imposed by the addition of these nucleotides to the mitochondrial suspension, in the presence of uncoupling agent and oligomycin, to inhibit ATPase. Under these conditions, mitochondrial CoASH/acetyl-CoA, NAD+/ NADH, and ADP/ATP ratios could be varied independently, and the effect on the interconversion of active and inactive pyruvate dehydrogenase could be studied. Decreases in both CoASH/acetyl-CoA and NAD+/NADH ratios were shown to be inhibitory to the steady state activity of pyruvate dehydrogenase, and this effect is described at three different ADP/ATP ratios and different concentrations of added MgCl2. A new steady state level of activity was achieved within 10 min of a change in either CoASH/acetyl-CoA or NAD+/NADH ratio; the rate of inactivation was much higher than the rate of reactivation under these conditions. Effects of CoASH/acetyl-CoA and NAD+/NADH may be additive but are still quantitatively lesser than the changes in activity of pyruvate dehydrogenase induced by changes in ADP/ATP ratio. The variation in activity of pyruvate dehydrogenase with ADP/ATP ratio is described in the absence of changes in the other two ratios, conditions which were not met in earlier studies which employed the oxidation of different substrates to generate changes in all three ratios.     

Measurement of mitochondrial and non-mitochondrial Ca2+ in isolated intact hepatocytes: a critical re-evaluation of the use of mitochondrial inhibitors.

Isolated rat hepatocytes treated with mitochondrial inhibitors FCCP or antimycin A release discrete amounts of Ca2+ in a Ca(2+)-free extracellular medium as revealed by changes in the absorbance of the Ca2+ indicator arsenazo III. The process is completed in 2 min and the amount of Ca2+ released is not affected by the type of the mitochondrial poison employed. The subsequent treatment with the cation ionophore A23187 causes a further release of Ca2+ that does not appear related to the specificity of the previous treatment with FCCP or antimycin A. Both FCCP and antimycin A cause a progressive loss of cellular ATP associated with a decrease in the ATP/ADP ratio from 6 to 2-1.5. However, this decrease does not significantly prevent 45Ca2+ accumulation in isolated liver microsomes. Moreover, the decrease of the ATP/ADP ratio to 1, does not promote a significant release of 45Ca2+ from 45Ca(2+)-preloaded microsomes. Finally, experiments with Fura-2-loaded hepatocytes reveal that agents specifically releasing Ca2+ from non-mitochondrial stores (vasopressin and 2,5-di-tert-butyl-1-4-benzohydroquinone) are still able to increase the cytosolic Ca2+ concentration in FCCP-treated cells. Taken together, these findings demonstrate that, in freshly isolated hepatocytes, FCCP specifically releases Ca2+ from mitochondrial stores without significantly affecting active Ca2+ sequestration in other cellular pools. For these reasons, FCCP can be used to release and quantitate mitochondrial Ca2+ in liver cells.     

Subcellular distribution of energy metabolites in the pre-ischaemic and post-ischaemic perfused working rat heart

Isolated working rat hearts were subjected to 20 min of global ischaemia and either 5 min or 15 min of reperfusion. The subcellular distribution of ATP, ADP, AMP, phosphocreatine and Pi were determined before and after ischaemia by the method of non-aqueous tissue fractionation. Ventricular function and the cytosolic, mitochondrial and ATPase-associated compartmentation of metabolites were measured. After 5 min of reperfusion, only 13 +/- 9% of the pre-ischaemic contractile function was restored compared to 67 +/- 8% after 15 min reperfusion. ATP was reduced in all cellular compartments after 5 min of reperfusion but was only decreased from pre-ischaemic values in the cytosolic compartment after 15 min of reperfusion (17.1 +/- 3.9 nmol/mg vs. 4.3 +/- 1.5 nmol/mg total protein; P less than 0.05). The mitochondrial [ATP]/[ADP] was reduced from a normal value of 4.36 to 1.79 after 5 min but recovered to 4.62 after 15 min of reperfusion. Most of the Pi was located in the mitochondria or with the ATPase fraction of the cell, with only 16% of the total Pi free in the cytosol. This study indicates that the capacity of the heart to recover function may be compromised during early reperfusion by a 59% increase in mitochondrial phosphate content and during late reperfusion by a reduced cytosolic/mitochondrial concentration ratio of both ATP (from 0.85 to 0.19) and phosphocreatine (from 3.9 to 1.24).     

Adenine nucleotides in foetal rat liver cells. Compartmentation and variation with age.

The digitonin method for the separation of cytosolic and mitochondrial fractions was applied to liver cells isolated from foetal rats. The cytosolic [ATP]/[ADP] ratio approximately doubles during the last 4 days of gestation, whereas the mitochondrial ratio remains constant. In the presence of oligomycin and added glucose, the cytosolic [ATP]/[ADP] ratio does not increase with age, but is still considerably higher than the mitochondrial ratio. Without added glucose, and when the glycogen content of foetal liver is still very low (more than 3 days before birth), the cytosolic [ATP]/[ADP] ratio in the presence of oligomycin becomes very low and equal to the mitochondrial ratio. It is concluded that the increasein the cytosolic [ATP]/[ADP] ratio during the last 4 days of gestation is solely due to enhanced mitochondrial activity in this period. Atractyloside and bongkrekic acid do not influence the O2 consumption, nor the [ATP]/[ADP] ratios in either compartment of foetal liver cells. Respiration of isolated foetal mitochondria, however, is strongly inhibited by both compounds. The implications of these findings are discussed.     

Cyclic AMP-induced Mg2+ release from rat liver hepatocytes, permeabilized hepatocytes, and isolated mitochondria.

The addition of norepinephrine, epinephrine, or forskolin to collagenase-dispersed rat liver hepatocytes increase cAMP and result in a 15% loss in total cell Mg2+ within 5 min. Conversely, carbachol and vasopressin induce a 10-15% increase of total cell Mg2+. Permeabilized hepatocytes also mobilize a large pool of Mg2+ when stimulated by ADP or cAMP. This stimulation is completely inhibited by atractyloside and bongkrekic acid, two different specific inhibitors of the mitochondrial adenine nucleotide translocase. cAMP directly mobilizes Mg2+ efflux from isolated rat liver mitochondria. 50 nM cAMP or 250 microM ADP induces in 5 min a mitochondrial loss of about 6 nmol of Mg2+/mg of protein and a stimulation of ATP efflux. The effect of cAMP is specific, is not reproduced by other cyclic or noncyclic nucleotides, and is inhibited by inhibitors of the adenine nucleotide translocase. These data indicate that cAMP is a messenger for a major mobilization of Mg2+ in hepatocytes. A major target for the effect of cAMP are mitochondria, which lose up to 20-25% of their total Mg2+ in 5 min, both within the cell and after isolation. Evidence is presented suggesting that the adenine nucleotide translocase is the target of the cAMP-dependent Mg2+ efflux and that cAMP may change the operation of the translocase. This, in turn, could change within the matrix the substrate of choice of the translocase from ATP to ATP.Mg.     

Effect of long-chain fatty acyl-CoA on mitochondrial and cytosolic ATP/ADP ratios in the intact liver cell.

The effect of long-chain acyl-CoA on subcellular adenine nucleotide systems was studied in the intact liver cell. Long-chain acyl-CoA content was varied by varying the nutritional state (fed and starved states) or by addition of oleate. Starvation led to an increase in the mitochondrial and a decrease in the cytosolic ATP/ADP ratio in liver both in vivo and in the isolated perfused organ as compared with the fed state. The changes were reversed on re-feeding glucose in liver in vivo or on infusion of substrates (glucose, glycerol) in the perfused liver, respectively. Similar changes in mitochondrial and cytosolic ATP/ADP ratios occurred on addition of oleate, but, importantly, not with a short-chain fatty acid such as octanoate. It is concluded that long-chain acyl-CoA exerts an inhibitory effect on mitochondrial adenine nucleotide translocation in the intact cell, as was previously postulated in the literature from data obtained with isolated mitochondria. The physiological relevance with respect to pyruvate metabolism, i.e. regulation of pyruvate carboxylase and pyruvate dehydrogenase by the mitochondrial ATP/ADP ratio, is discussed.     

Metabolic control of mitochondrial properties by adenine nucleotide translocator determines palmitoyl-CoA effects. Implications for a mechanism linking obesity and type 2 diabetes

Inhibition of the mitochondrial adenine nucleotide translocator (ANT) by long-chain acyl-CoA esters has been proposed to contribute to cellular dysfunction in obesity and type 2 diabetes by increasing formation of reactive oxygen species and adenosine via effects on the coenzyme Q redox state, mitochondrial membrane potential (Deltapsi) and cytosolic ATP concentrations. We here show that 5 microm palmitoyl-CoA increases the ratio of reduced to oxidized coenzyme Q (QH(2)/Q) by 42 +/- 9%, Deltapsi by 13 +/- 1 mV (9%), and the intramitochondrial ATP/ADP ratio by 352 +/- 34%, and decreases the extramitochondrial ATP/ADP ratio by 63 +/- 4% in actively phosphorylating mitochondria. The latter reduction is expected to translate into a 24% higher extramitochondrial AMP concentration. Furthermore, palmitoyl-CoA induced concentration-dependent H(2)O(2) formation, which can only partly be explained by its effect on Deltapsi. Although all measured fluxes and intermediate concentrations were affected by palmitoyl-CoA, modular kinetic analysis revealed that this resulted mainly from inhibition of the ANT. Through Metabolic Control Analysis, we then determined to what extent the ANT controls the investigated mitochondrial properties. Under steady-state conditions, the ANT moderately controlled oxygen uptake (control coefficient C = 0.13) and phosphorylation (C = 0.14) flux. It controlled intramitochondrial (C = -0.70) and extramitochondrial ATP/ADP ratios (C = 0.23) more strongly, whereas the control exerted over the QH(2)/Q ratio (C = -0.04) and Deltapsi (C = -0.01) was small. Quantitative assessment of the effects of palmitoyl-CoA showed that the mitochondrial properties that were most strongly controlled by the ANT were affected the most. Our observations suggest that long-chain acyl-CoA esters may contribute to cellular dysfunction in obesity and type 2 diabetes through effects on cellular energy metabolism and production of reactive oxygen species.     

Calcium stimulates ATP-Mg/Pi carrier activity in rat liver mitochondria

Adenine nucleotide transport over the carboxyatractyloside-insensitive ATP-Mg/Pi carrier was assayed in isolated rat liver mitochondria with the aim of investigating a possible regulatory role for Ca2+ on carrier activity. Net changes in the matrix adenine nucleotide content (ATP + ADP + AMP) occur when ATP-Mg exchanges for Pi over this carrier. The rates of net accumulation and net loss of adenine nucleotides were inhibited when free Ca2+ was chelated with EGTA and stimulated when buffered [Ca2+]free was increased from 1.0 to 4.0 microM. The unidirectional components of net change were similarly dependent on Ca2+; ATP influx and efflux were inhibited by EGTA in a concentration-dependent manner and stimulated by buffered free Ca2+ in the range 0.6-2.0 microM. For ATP influx, increasing the medium [Ca2+]free from 1.0 to 2.0 microM lowered the apparent Km for ATP from 4.44 to 2.44 mM with no effect on the apparent Vmax (3.55 and 3.76 nmol/min/mg with 1.0 and 2.0 microM [Ca2+]free, respectively). Stimulation of influx and efflux by [Ca2+]free was unaffected by either ruthenium red or the Ca2+ ionophore A23187. Calmodulin antagonists inhibited transport activity. In isolated hepatocytes, glucagon or vasopressin promoted an increased mitochondrial adenine nucleotide content. The effect of both hormones was blocked by EGTA, and for vasopressin, the effect was blocked also by neomycin. The results suggest that the increase in mitochondrial adenine nucleotide content that follows hormonal stimulation of hepatocytes is mediated by an increase in cytosolic [Ca2+]free that activates the ATP-Mg/Pi carrier.     

Evidence that the flux control coefficient of the respiratory chain is high during gluconeogenesis from lactate in hepatocytes from starved rats. Implications for the hormonal control of gluconeogenesis and action of hypoglycaemic agents.

1. Increasing concentrations of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), a mild respiratory-chain inhibitor [Halestrap (1987) Biochim. Biophys. Acta 927, 280-290], caused progressive inhibition of glucose production from lactate + pyruvate by hepatocytes from starved rats incubated in the presence or absence of oleate and gluconeogenic hormones. 2. No significant changes in tissue ATP content were observed, but there were concomitant decreases in ketone-body output and cytochrome c reduction and increases in NADH fluorescence and the ratios of [lactate]/[pyruvate] and [beta-hydroxybutyrate]/[acetoacetate]. 3. The inhibition by DCMU of palmitoylcarnitine oxidation by isolated liver mitochondria was used to calculate a flux control coefficient of the respiratory chain towards gluconeogenesis. In the presence of 1 mM-oleate, the calculated values were 0.61, 0.39 and 0.25 in the absence of hormone and in the presence of glucagon or phenylephrine respectively, consistent with activation of the respiratory chain in situ as previously suggested [Quinlan & Halestrap (1986) Biochem. J. 236, 789-800]. 4. Cytoplasmic oxaloacetate concentrations were shown to decrease under these conditions, implying inhibition of pyruvate carboxylase. 5. Inhibition of gluconeogenesis from fructose and dihydroxyacetone was also observed with DCMU and was accompanied by an increased output of lactate + pyruvate, suggesting that activation of pyruvate kinase was occurring. With the latter substrate, measurements of tissue ADP and ATP contents showed that DCMU caused a small fall in [ATP]/[ADP] ratio. 6. Two inhibitors of fatty acid oxidation, pent-4-enoate and 2-tetradecylglycidate, were shown to abolish and to decrease respectively the effects of hormones, but not valinomycin, on gluconeogenesis from lactate + pyruvate, without changing tissue ATP content. 7. It is concluded that the hormonal increase in mitochondrial matrix volume stimulates fatty acid oxidation and respiratory-chain activity, allowing stimulation of pyruvate carboxylation and thus gluconeogenesis to occur without major changes in [ATP]/[ADP] or [NADH]/[NAD+] ratios. 8. The high flux control coefficient of the respiratory chain towards gluconeogenesis may account for the hypoglycaemic effect of mild respiratory-chain inhibitors.     

Brown adipose tissue mitochondria: recoupling caused by substrate level phosphorylation and extramitochondrial adenosine phosphates.

1. Uncoupled oxidative phosphorylation in isolated guinea pig brown-adipose-tissue mitochondria is reflected by a low phosphorylation state of adenosine phosphates in the mitochondrial matrix and in the extramitochondrial space during oxidation of succinate or glycerol 1-phosphate in the presence of serum albumin and 100 muM ADP. Recoupling of respiration and phosphorylation in the mitochondria is indicatdd by a dramatic increase in the phosphorylation state of adenine nucleotides in both compartments, when substrates inducing substrate level phosphorylation are respired. In this case ATP/ADP ratios in the extramitochondrial compartment are 10-15 times higher than in the mitochondrial matrix. 2. Recoupling mediated by substrate level phosphorylation depends on the presence of extramitochondrial adenosine phosphate and on intact adenine nucleotide translocation. In the presence of substrate level phosphorylation the amount of extramitochondrial ADP required to restore energy coupling can be extremely low (20 muM ADP or 10 nmol ADP/mg mitochondrial protein respectively). If substrate level phosphorylation is prevented by rotenone or in the presence of atractyloside, 20-50 times higher amounts of extramitochondrial adenine nucleotides are necessary to cause coupled oxidative phosphorylation. The recoupling effect of ATP is significantly stronger than that of ADP. 3. GDP (100 muM) causes a rapid increase of the ATP/ADP ratio in both compartments which is independent of substrate level phosphorylation as well as of the extramitochondrial adenosine phosphate concentration and the adenine nucleotide carrier. 4. The amount of extramitochondrial adenosine phosphate in guinea pig brown-adipose-tissue (18 nmol/mg mitochondrial protein or 2.5 mM respectively) would suffice for recoupling of oxidative phosphorylation mediated by substrate level phosphorylation under conditions in vitro; this suggests that substrate level phosphorylation is of essential importance in brown fat in vivo with respect to energy conditions in the tissue during different states of thermogenesis.     

Role of intramitochondrial pH in the energetics and regulation of mitochondrial oxidative phosphorylation.

The dependence of ATP synthesis coupled to electron transfer from 3-hydroxy-butyrate (3-OH-B) to cytochrome c on the intramitochondrial pH (pHi) was investigated. Suspensions of isolated rat liver mitochondria were incubated at constant extramitochondrial pH (pHe) with ATP, ADP, Pi, 3-OH-B, and acetoacetate (acac) (the last two were varied to maintain [3-OH-B]/[acac] constant), with or without sodium propionate to change the intramitochondrial pH. Measurements were made of the steady-state water volume of the mitochondrial matrix, transmembrane pH difference, level of cytochrome c reduction, concentration of metabolites and rate of oxygen consumption. For each experiment, conditions were used for which transmembrane pH was near maximal and minimal values and the measured extramitochondrial [ATP], [ADP], and [Pi] were used to calculate log[ATP]/[ADP][Pi]. When [3-OH-B]/[acac] and [cyt c2+]/[cyt c3+] were constant, and pHi was decreased from approx. 7.7 to 7.2, log [ATP]/[ADP][Pi] at high pHi was significantly (P less than 0.02) greater than at low pHi. The mean slope (delta log [ATP]/[ADP][Pi] divided by the change in pHi) was 1.08 +/- 0.15 (mean +/- S.E.). This agrees with the slope of 1.0 predicted if the energy available for ATP synthesis is dependent upon the pH at which 3-hydroxybutyrate dehydrogenase operates, that is, on the pH of the matrix space. The steady-state respiratory rate and reduction of cytochrome c were measured at different pHi and pHe values. Plots of respiratory rate vs.% cytochrome c reduction at different intra- and extramitochondrial pH values indicated that the respiratory rate is dependent upon pHi and not on pHe. This implies that the matrix space is the source of protons involved in the reduction of oxygen to water in coupled mitochondria.