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.

Compartmentation of high-energy phosphates in resting and working rat skeletal muscle.

The subcellular distribution of high-energy phosphates in various types of skeletal muscle of the rat was analysed by subfractionation of tissues in non-aqueous solvents. Different glycolytic and oxidative capacities were calculated from the ratio of phosphoglycerate kinase and citrate synthase activities, ranging from 25 in m. soleus to 130 in m. tensor fasciae latae. In the resting state, the subcellular contents of ATP, creatine phosphate and creatine were similar in m. soleus, m. vastus intermedius, m. gastrocnemius and m. tensor fasciae latae but, significantly, a higher extramitochondrial ADP-content was found in m. soleus. A similar observation was made in isometrically and isotonically working m. gastrocnemius. The extramitochondrial, bound ADP accounted fully for actin-binding sites in resting fast-twitch muscles, but an excess of bound ADP was found in m. soleus and working m. gastrocnemius. The amount of non-actin-bound ADP reached maximal values of approx. 1.2 nmol/mg total protein. It could not be enhanced further by prolonged isotonic stimulation or by increased isometric force development. It is suggested that non-actin-bound ADP is accounted for by actomyosin-ADP complexes generated during the contraction cycle. Binding of extramitochondrial ADP to actomyosin complexes in working muscles thus acts as a buffer for cytosolic ADP in addition to the creatine system, maintaining a high cytosolic phosphorylation potential also at increasing rates of ATP hydrolysis during muscle contraction.

The role of the mitochondrial 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, carboxyatractyloside) 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.

Effects of hypoxia and fatty acids on the distribution of metabolites in rat heart.

The effects of exogenous fatty acids and hypoxia on cardiac energy metabolism were studied by measuring mitochondrial and cytosolic adenine nucleotides as well as CoA and carnitine esters using a tissue fractionation technique in non-aqueous solvents. During normoxia, the administration of 0.5 mM palmitate caused a considerable increase in acyl-CoA and acylcarnitine, particularly in mitochondria. High-energy phosphates, however, were only slightly altered. A 90 min low-flow hypoxia caused a dramatic increase in mitochondrial acyl esters. The mitochondrial ATP content decreased significantly, while the cytosolic concentration was only slightly diminished, suggesting an inhibition of mitochondrial adenine nucleotide translocation by long-chain acyl-CoA. Addition of palmitate during hypoxia amplified hypoxic damage and reduced adenine nucleotides in both compartments considerably, while fatty acid metabolites were only slightly affected. In presence of an inhibitor of fatty acid oxidation (BM 42.304), the fatty-acid-induced acceleration of cardiac injury was prevented. Since BM 42.304 decreased mitochondrial acylcarnitine and increased the cytosolic concentration significantly, BM 42.304 was presumed to inhibit mitochondrial acylcarnitine translocase. However, a causal relationship between lipid metabolites and ischemic damage seemed unlikely.

Determination of mitochondrial creatine kinase fluxes in intact heart mitochondria using 31P-saturation transfer nuclear magnetic resonance spectroscopy.

Forward (-->ATP) and reverse (-->CrP) fluxes through the creatine kinase reaction were determined in isolated rat and bovine heart mitochondria and with soluble MM-CK from rabbit skeletal muscle, using 31P-saturation transfer NMR. With soluble MM-CK forward and reverse fluxes were identical in the absence and presence of BSA or rat liver mitochondria. Addition of liver mitochondria decreased fluxes with increasing mitochondria concentration. The fluxf/Vmax(f) ratio was 0.006 with 10 mg BSA and 0.04 with 10 mg rat liver mitochondria, respectively. With heart mitochondria, fluxr was considerably higher than fluxf and the fluxf/Vmax(f) ratio was 1.7 for rat heart and 0.22 for bovine heart. It is concluded that in the presence of isolated mitochondria, the flux through the creatine kinase is driven by the mitochondrial ATP-ADP turnover. Therefore the fluxf/Vmax(f) ratio is highest for rat heart mitochondria with a high ATP-ADP turnover, intermediate for bovine heart mitochondria and low for MM-CK in the presence of liver mitochondria. It is lowest with MM-CK alone, where the creatine kinase reaction is at equilibrium and external ATP-ADP turnover is absent. The higher reverse than forward fluxes of mitochondrial creatine kinase determined at steady state by saturation transfer NMR, are caused mainly by a high ATP<-->Pi exchange in heart mitochondria preparations, having a high ATPase activity, compared to liver mitochondria.

In vitro determination of creatine kinase substrate fluxes using 31P-nuclear magnetic resonance.

Forward (kf) and reverse (kr) rate constants and the corresponding flux rates of the creatine kinase catalysed reaction between creatine phosphate (CrP) and adenosine triphosphate (ATP); CrP + ADP<-->kf kr ATP + Cr were measured in vitro at 295 K. Both rate constants were determined using magnetic resonance saturation transfer techniques. To study the dependence of kr and kf on the creatine kinase concentration, the creatine kinase activity was varied from 2400 to 75 U.ml-1. At equilibrium and high creatine kinase activities, the forward to reverse flux rate ratios are close to 1. A dispersion in the reaction rate constants was observed at activities < or = 600 U.ml-1. We measured kr > kf for all enzyme activities below 600 U.ml-1. This observation could partially be explained by the presence of ATPase contamination in the enzyme. These findings are relevant for the in vivo studies of creatine kinase activity in the presence of multi-site phosphate exchange in cellular ATP-pools. As mitochondrial creatine kinase is not in equilibrium these results are also of interest in this area.

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.

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.

Stimulation of oxygen consumption following addition of lipid substrates in liver and skeletal muscle from rats fed a high-fat diet.

We studied hepatic and skeletal muscle metabolic activity in rats fed a high-fat diet. Rats were fed a low-fat or high-fat diet for 15 days. At the end of the experimental period, full energy-balance determinations together with serum free triiodothyronine (FT3), leptin, and free fatty acid (FFA) measurements were performed. In addition, we assessed fatty acid-stimulated oxygen consumption in perfused liver and in skeletal muscle homogenate. Rats fed a high-fat diet showed a significant increase in energy intake but no variation in body energy gain, due to a significant increase in energy expenditure. Serum FT3 and FFA levels significantly increased in rats fed a high-fat diet versus rats fed a low-fat diet, while no variation was found in serum leptin levels. Perfused livers and skeletal muscle homogenates from rats fed a high-fat diet exhibited a significant increase in fatty acid-stimulated oxygen consumption. Our results suggest that the enhanced fatty acid oxidation rates in liver and skeletal muscle contribute to the maintenance of fat balance in response to increased fat intake, preventing excess fat deposition.

[Potential improvements in medical education as retrospectively evaluated by candidates for specialist examinations]. / Verbesserungspotenzial des Medizinstudiums aus retrospektiver Sicht von Facharztprüflingen.

BACKGROUND AND OBJECTIVE: As part of the new regulations for licensing doctors there have been numerous attempts at reform by many medical faculties to consider interdisciplinary linkage of the curriculum with emphasis on teaching of small groups of students. This study was undertaken to help answer the question of how much weight should be given to the various subjects and what resources are needed for any reformed curriculum and what key areas of competence need to be given greater importance. METHODS: 1029 candidates of specialist examinations of the Medical Council of North-Rhine in 2002 and 2003 filled in questionnaires to evaluate retrospectively the actual relevance of individual preclinical and clinical subjects, courses and areas of practical competence to their further medical education and related potentials for improvement in their studies. The participants were from 5 medical faculties in the North-Rhine area of Germany. They were also asked about methods of examination that were effective in aiding their learning behaviour. RESULTS: Those answering the questionnaire considered especially chemistry and physics as well as environmental, occupational and forensic medicine, bio-mathematics, radiotherapy and nuclear medicine among the less relevant subjects. On the other hand, anatomy, physiology, internal medicine, pharmacology and surgery were considered especially relevant. CONCLUSION: The greatest deficiencies in most of the medical curricula as taught in the North-Rhine medical courses are in the areas of competence in communication and practical clinical skills. Members of this group also pleaded for an increased use of standardized objective structured clinical examinations (OSCE).

Long-term and short-term changes in mitochondrial parameters by thyroid hormones.

In the hyperthyroid state, delta psi m, delta pHm and therefore delta p are increased in rat liver. An enhanced delta p accords with a higher energy output. The subcellular distribution of adenine nucleotides in different thyroid states does not reflect the driving force for mitochondrial adenine-nucleotide translocase (that is delta psi m). Therefore, a change in delta psi m cannot be solely responsible for the postulated stimulation of adenine-nucleotide transport by THs. This is also the case for the changes in delta pHm, and in the subcellular distribution of malate, 2-oxoglutarate and glutamate, that are observed under the influence of THs. T3 induces calcium influx into the liver cell within minutes. It increases respiration and gluconeogenesis with the same kinetics. Therefore, it is suggested that, as with glucagon and vasopressin, calcium is the mediator of these changes. The delta p is increased with T3 and glucagon treatment but not with vasopressin. The changes in delta psi m and delta pHm appear to be the result of the individual actions of these hormones on ATP-consuming and ATP-producing reactions. The delta psi p is only increased with T3 treatment. This is related to the different mechanisms of enhancing intracellular calcium that are used by vasopressin, glucagon and T3.