Sibylle Soboll

Thyroid hormone action on mitochondrial energy transfer.

II. Thyroid hormone signalling pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 A. Binding sites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 1. Plasma membrane binding sites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 2. Cytosolic binding sites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 3. Mitochondrial binding sites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 4. Nuclear binding sites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 5. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 B. Nuclear signalling pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 1. Regulation of transcription of mitochondrial proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 2. Regulation of transcription of proteins involved in intracellular signalling . . . . . . . . . . . . . . 5 C. Extranuclear signalling pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 1. Action at the plasma membrane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 2. Action at the mitochondrial membrane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Influence of fatty acids on energy metabolism. 1. Stimulation of oxygen consumption, ketogenesis and CO2 production following addition of octanoate and oleate in perfused rat liver.

: Changes in metabolic rates (oxygen consumption, ketogenesis, 14CO2 production from labelled fatty acids, glycolysis) following the addition of octanoate or oleate were studied in isolated livers from fed and starved rats perfused with Krebs-Henseleit bicarbonate buffer in a non-recirculating system. The following results were obtained. The infusion of fatty acids caused a large increase in the rate of oxygen consumption. The effect was greater with octanoate than with oleate and was half-maximal with fatty acid concentrations (free plus albumin bound) around 0.1 mM. The effects of oleate were only partially suppressed when the perfusate contained albumin concentrations near the physiological range. When fatty acids were oxidized at high rates, the glycolytic rate was diminished by 50%. The increase in oxygen consumption could not be explained fully by the increased ATP demand for fatty acid metabolism or by a compensation for the diminished extramitochondrial ATP generation. In the presence of phenylalkyl oxirane carboxylic acid, an inhibitor of the transport of long-chain acyl-CoA derivates into the mitochondria, ketogenesis and 14CO2 production from labelled oleate were strongly inhibited, whereas the increase in oxygen consumption was only slightly affected. In the presence of antimycin A, the increase in oxygen consumption due to fatty acids was totally abolished. Following pretreatment of rats with ciprofibrate (induction of enzymes for peroxisomal beta-oxidation of long-chain fatty acids), ketogenesis (but not 14CO2 production) from oleate was enhanced threefold. The increase in oxygen consumption, however, was not affected. In conclusion, the increase in hepatic oxygen consumption due to addition of fatty acids reflects a mitochondrial process; it is, in part, independent of the ATP demand of the cell. An uncoupling-like effect of fatty acids on the respiratory chain and its possible physiological significance in ketogenesis are discussed.

Regulation of the degree of coupling of oxidative phosphorylation in intact rat liver.

The degree of coupling of oxidative phopshorylation q was determined in isolated perfused livers and in livers in vivo from fed and fasted rats. This determination of q was based on a simple nonequilibrium-thermodynamic representation of the major reactions of cytosolic adenine nucleotides, and made use of the measured cytosolic concentrations of adenine nucleotides, phosphate, and lactate/pyruvate ratios in extracted livers. The deviations of the measured values from the theoretically predicted ones at different mass action ratios of the adenylate kinase reaction showed that the basic assumptions of the model, including linearity between flows and thermodynamic forces, were fulfilled in intact liver within the experimental error. The degree of coupling was higher in livers from fed rats than in livers from fasted rats. In particular, the determined values of q were close to the theoretical degrees of coupling qecp and qecf which allow maximization of output power and output flow of oxidative phosphorylation for fed and fasted states, respectively, at optimal efficiency and minimal energy costs. This finding indicates that conductance matching between the load and phosphorylation is fulfilled in vivo. Moreover, it was found that fatty acids lower the degree of coupling in a concentration-dependent manner. This suggested that in livers in the fasted state q is decreased due to elevated fatty-acid levels. Thus fatty acids could act as metabolic regulators of the degree of coupling, enabling the cell to optimize efficiency of oxidative phosphorylation under different metabolic regimes.

Regulation of energy metabolism in liver

Energy metabolism in liver has to cope with the special tasks of this organ in intermediary metabolism. Main ATP-generating processes in the liver cell are the respiratory chain and glycolysis, whereas main ATP-consuming processes are gluconeogenesis, urea synthesis, protein synthesis, ATPases and mitochondrial proton leak. Mitochondrial respiratory chain in the intact liver cell is subject to control mainly by substrate (hydrogen donors, ADP, oxygen) transport and supply and proton leak/slip. Whereas hormonal control is mainly on substrate supply to mitochondria, proton leak/slip is supposed to play an important role in the modulation of the efficiency of oxidative phosphorylation.

Influence of fatty acids on energy metabolism. 2. Kinetics of changes in metabolic rates and changes in subcellular adenine nucleotide contents and pH gradients following addition of octanoate and oleate in perfused rat liver

The uncoupling-like effect of fatty acids [ Scholz , R., Schwabe , U., and Soboll , S. (1984) Eur. J. Biochem. 141, 223-230] was further substantiated in experiments with perfused rat livers by two ways: firstly the kinetics of changes in metabolic rates (oxygen consumption, ketogenesis, fatty acid oxidation) were analysed; secondly subcellular contents of adenine nucleotides and pH gradients across the mitochondrial membrane were determined following fractionation of freeze-fixed and dried tissues in non-aqueous solvents. The following results were obtained. The relaxation kinetics of the increase in oxygen consumption following fatty acid infusion revealed two components, a rapid one with a half-time around 10 s and a slow one with a half-time of more than 100 s. The rapid component was similar to the kinetics of fatty acid oxidation (ketogenesis and 14CO2 production from labelled fatty acids) whereas the half-time of the slow component was in the range of half-times observed with the increase in oxygen consumption following addition of carbonylcyanide p-trifluoromethoxyphenylhydrazone. In the presence of fatty acids, the cytosolic ATP concentrations and ATP/ADP ratios decreased, whereas the corresponding parameters for the mitochondrial space were either increased (oleate) or decreased (octanoate). The effects of oleate were dependent on the albumin concentrations in the perfusate. The normally large difference between cytosolic and mitochondrial ATP/ADP ratios became smaller. Similar observations were obtained with uncoupling agents. The pH gradient across the mitochondrial membrane as calculated from the subcellular distribution of 5,5 dimethyl[2-14C]oxazolidine-2,4-dione was inversed following the addition of both carbonylcyanide p-trifluoromethoxyphenylhydrazone and fatty acids, i.e. the mitochondrial matrix became more acidic than the cytosol. The pH gradient was not affected when oleate was added in the presence of high albumin concentrations. The data support the hypothesis that the increase in hepatic oxygen consumption due to octanoate or oleate is, in part, caused by a mechanism similar to uncoupling of oxidative phosphorylation. This mechanism seems not to be an artifact of isolated systems; it may be of physiological importance for processes in which reducing equivalents are removed independently of the ATP demand of the hepatocyte.

The role of the mitochondriial creatine kinase system for myocardial function during ischemia and reperfusion

The subcellular distribution of ATP, ADP, creatine phosphate and creatine was studied in normoxic control, isoprenaline-stimulated and potassium-arrested guinea-pig hearts as well as during ischemia and after reperfusion. The mitochondrial creatine phosphate/creatine ratio was closely correlated to the oxidative activity of the hearts. This was interpreted as an indication of a close coupling of mitochondrial creatine kinase to oxidative phosphorylation. To further investigate the functional coupling of mitochondrial creatine kinase to oxidative phosphorylation, rat or guinea-pig heart mitochondria were isolated and the mass action ratio of creatine kinase determined at active or inhibited oxidative phosphorylation or in the presence of high phosphate, conditions which are known to change the functional state of the mitochondrial enzyme. At active oxidative phosphorylation the mass action ratio was one-third of the equilibrium value whereas at inhibited oxidative phosphorylation (N2, oligomycin, car☐yatractyloside) or in the presence of high phosphate, the mass action ratio reached equilibrium values. These findings show that oxidative phosphorylation is essential for the regulation of the functional state of mitochondrial creatine kinase. The functional coupling of the mitochondrial creatine kinase and oxidative phosphorylation indicated from the correlation of mitochondrial creatine phosphate/creatine ratios with the oxidative activity of the heart in situ as well as from the deviation of the mass action ratio of the mitochondrial enzyme from creatine kinase equilibrium at active oxidative phosphorylation in isolated mitochondria is in accordance with the proposed operation of a creatine shuttle in heart tissue.

Control of energy metabolism by glucagon and adrenaline in perfused rat liver

Changes in subcellular distribution of adenine nucleotides, mitochondrial/cytosolic proton gradients, rates of respiration, gluconeogenesis (fasted state) and glycogenolysis (fed state) were studied in isolated perfused rat livers following addition of glucagon (10−8 M) or adrenaline (10−7 M). Glucagon increased the gradient in all states. The cytosolic ATP/ADP ratio was increased in the fasted but decreased in the fed state which is consistent with a diminished futile cycling in gluconeogenesis (fasted state) or a decreased glycolytic rate (fed state). Adrenaline caused an increase in the proton gradient and the mitochondrial ATP/ADP ratio. The two effects are attributed to increased calcium entry into the matrix space.

UPTAKE OF CREATINE PHOSPHATE INTO HEART MITOCHONDRIA : A LEAK IN THE CREATINE SHUTTLE

CrP uptake into isolated rat heart mitochondria was studied using silicone oil centrifugation. Further, the involvement of the mitochondrial adenine nucleotide translocase was examined by measuring CrP accumulation in mitochondria in the presence of substrates and inhibitors of the ATP/ADP-carrier and by investigating uptake kinetics in liposomes reconstituted with purified bovine heart adenine nucleotide translocase protein. CrP is accumulated in the matrix space of isolated rat heart mitochondria and mitoplasts. The uptake is inhibited by carboxyatractyloside, a specific inhibitor of the mitochondrial adenine nucleotide translocase, and by ADP, phosphoenolpyruvate, 3-phosphoglycerate and pyrophosphate, compounds which are able to bind to the carrier. It is not inhibited when the mitochondrial membrane potential is decreased. CrP is transported into reconstituted liposomes at a rate which is about 3 orders of magnitude lower than the rate for ATP uptake. The transport is sensitive to temperature change and to carboxyatractyloside. It is concluded that CrP is specifically taken up by heart mitochondria via the mitochondrial adenine nucleotide translocase. The transport in mitochondria in situ is facilitated by the close local and functional interaction of the mitochondrial creatine kinase and the adenine nucleotide translocase within contact sites between inner and outer mitochondrial membrane. A certain amount of CrP synthesized by the mitochondrial creatine kinase thus escapes its usage at cytosolic energy consuming processes.

Acetyl-L-carnitine treatment stimulates oxygen consumption and biosynthetic function in perfused liver of young and old rats

Abstract: The effect of treatment with acetyl-L-carnitine on hepatic mitochondrial respiration and biosynthetic function in perfused liver from young (90 days) and old (22-24 months) rats was studied. Rats were given a 1.5% (w/v) solution of acetyl-L-carnitine in their drinking water for 1 month and oxygen consumption together with the rate of gluconeogenesis, urea synthesis, and ketogenesis with and without added substrates were measured in perfused liver. Mitochondrial oxygen consumption was also assessed in liver homogenate and isolated mitochondria to determine the maximal capacity for oxidative phosphorylation. Acetyl-L-carnitine treatment almost completely restored the age-dependent decline in oxygen consumption, gluconeogenesis, urea synthesis, and ketogenesis found in perfused liver of old rats to the levels found in young rats. In addition, acetyl-L-carnitine treatment increased oxygen consumption and biosynthetic function in perfused liver from young rats. After acetyl-L-carnitine treatment, we found detectable 3-oxoacyl-CoA-transferase activity associated with a consumption of ketone bodies in young and old rats. Finally, oxygen consumption measured in homogenate and isolated mitochondria did not change with age and acetyl-L-carnitine treatment. Our results show that in perfused liver, acetyl-L-carnitine treatment slows the age-associated decline in mitochondrial respiration and biosynthetic function. In addition, treatment of young rats with acetyl-L-carnitine has a stimulating effect on liver metabolism, probably through an increase in ATP production.

Steady state changes in mitochondrial electrical potential and proton gradient in perfused liver from rats fed a high fat diet

In this work the protonmotive force (Δp), as well as the subcellular distribution of malate, ATP, and ADP were determined in perfused liver from rats fed a low fat or high fat diet, using density gradient fractionation in non acqueous solvents.Rats fed a high fat diet, despite an enhanced hepatic oxygen consumption, exhibit similar Δp to that found in rats fed a low fat diet, but when we consider the two components of Δp, we find a significant decrease in mitochondrial/cytosolic pH difference (ΔpHm) and a significant increase in mitochondrial membrane potential (ΔΨm) in rats fed a high fat diet compared to rats fed a low fat diet, which tend to compensate each other. In rats fed a high fat diet the concentration ratio of malate and ATP/ADP does not reflect the changes in ΔpHm and ΔΨm, which represent the respective driving force for their transport.The findings are in line with an increase in substrate supply to the respiratory chain which is, however, accompanied by a higher energy turnover in livers from HFD rats. By this way the liver could contribute to the lack of weight gain from the high caloric intake in HFD rats.