The role of brown adipose tissue in total energy balance and cold-induced thermogenesis was studied. Mice expressing mitochondrial uncoupling protein 1 (UCP-1) from the fat-specific aP2 gene promoter (heterozygous and homozygous aP2 -Ucp transgenic mice) and their nontransgenic C57BL6/J littermates were used. The transgenic animals are resistant to obesity induced by a high-fat diet, presumably due to ectopic synthesis of UCP-1 in white fat. These animals exhibited atrophy of brown adipose tissue, as indicated by smaller size of brown fat and reduction of its total UCP-1 and DNA contents. Norepinephrine-induced respiration (measured in pentobarbital sodium-anesthetized animals) was decreased proportionally to the dosage of the transgene, and the homozygous (but not heterozygous) transgenic mice exhibited a reduction in their capacity to maintain body temperature in the cold. Our results indicate that the role of brown fat in cold-induced thermogenesis cannot be substituted by increased energy expenditure in other tissues.

The obesogenic effect of a high-fat (HF) diet is counterbalanced by stimulation of energy expenditure and lipid oxidation in response to a meal. The aim of this study was to reveal whether muscle nonshivering thermogenesis could be stimulated by a HF diet, especially in obesity-resistant A/J compared with obesity-prone C57BL/6J (B/6J) mice. Experiments were performed on male mice born and maintained at 30 degrees C. Four-week-old mice were randomly weaned onto a low-fat (LF) or HF diet for 2 wk. In the A/J LF mice, cold exposure (4 degrees C) resulted in hypothermia, whereas the A/J HF, B/6J LF, and B/6J HF mice were cold tolerant. Cold sensitivity of the A/J LF mice was associated with a relatively low whole body energy expenditure under resting conditions, which was normalized by the HF diet. In both strains, the HF diet induced uncoupling protein-1-mediated thermogenesis, with a stronger induction in A/J mice. Only in A/J mice: 1) the HF diet augmented activation of whole body lipid oxidation by cold; and 2) at 30 degrees C, oxygen consumption, total content, and phosphorylation of AMP-activated protein kinase (AMPK), and AICAR-stimulated palmitate oxidation in soleus muscle was increased by the HF diet in parallel with significantly increased leptinemia. Gene expression data in soleus muscle of the A/J HF mice indicated a shift from carbohydrate to fatty acid oxidation. Our results suggest a role for muscle nonshivering thermogenesis and lipid oxidation in the obesity-resistant phenotype of A/J mice and indicate that a HF diet could induce thermogenesis in oxidative muscle, possibly via the leptin-AMPK axis.

Experimental Physiology : Translation and IntegrationCells with high and fluctuating energy demands (e.g. muscle tissue) require an effective system for metabolic control and energy transfer. The most effective system, integrating energy metabolism into one efficiently regulated metabolic network, is the creatine kinase (CK) shuttle (for review see Walliman et al. 1992;Saks et al. 1996). Creatine kinase (EC 2.7.3.2) controls the near-equilibrium (Kushmerick, 1983) CK reaction:in heart, skeletal muscle, brain and smooth muscle.The spatial organization of creatine kinase isoenzymes has been long recognized, and striated muscle cells are the best example of energy metabolism compartmentation. The CK isoenzymes are localized into energy-producing and energy-utilizing sites, where they are functionally coupled with ATP synthesis (mitochondria, cytosol) or ATPconsuming processes (myofibrils, sarcoplasmic reticulum). This organization of the CK system ensures the regulation of local concentrations of ADP and ATP, maintenance of the optimal ATP/ADP ratio, regulation of adenylate nucleotide fluxes and protection of the adenine nucleotides cellular pool from degradation.It can be seen from eqn (1) that first, the position of the CK reaction equilibrium should be affected by cytoplasmic pH, and second, the CK reaction evidently deviates from equilibrium, and its regulation should be described in terms of non-equilibrium thermodynamics (Mejsnar et al. 1992;Maršík & Mejsnar, 1994). The regulation can be realized by conformational changes of the CK molecule, when its reactive 'closed' conformation is not achieved merely by the substrate-induced energy-minimizing principle (Mejsnar et al. 2002).Stated in another way, any ATPase system that evokes a unidirectional net reverse CK flux towards ATP, by the splitting of ATP will shift the CK reaction out of equilibrium. The functional coupling of myosin ATPase and myofibrillar CK by substrate channelling (ArrioDupont, 1988;Gregor et al. 1999), which is defined as direct transfer of ATP between active sites of these enzymes, emphasizes the key role of the phosphocreatine/creatine Substrate channelling in a creatine kinase system of rat skeletal muscle under various pH conditions The aim of this study was to evaluate myofibrillar creatine kinase (CK) activity and to quantify the substrate channelling of ATP between CK and myosin ATPase under different pH conditions within the integrity of myofibrils. A pure myofibrillar fraction was prepared using differential centrifugation. The homogeneity of the preparation and the purity of the fraction were confirmed microscopically and by enzymatic assays for contaminant enzyme activities. The specific activity of myofibrillar CK reached 584 ± 33 nmol PCr min _1 mg _1 at pH 6.75. Two methods were used to detect CK activity: (1) measurement of direct ATP production, and (2) measurement of PCr consumption. This method of evaluation has been tested in experiments with isolated creatine kinase. No discrepancy in CK activity between the methods was ...

Creatine kinase (CK) (E.C. 2.7.3.2) buffers cellular ATP concentration during fluctuating ATP turnover. Muscle cytosolic CK isoform interacts with various subcellular structures where it is functionally coupled with relevant ATPases. However, how this interaction affects its activity is not known. We have therefore studied the interaction of CK with myofibrils and the role of different conformational states of CK molecule induced by ATP, phosphocreatine, ADP and the ATP-creatine pair. Purified rabbit psoas myofibrils with CK specific activity of 0.4+/-0.02 IU/mg were used. The exchange rates between the myofibrillar M-band and its surroundings were measured with fluorofore conjugated CK (IAF) by the Fluorescence Lost in Photobleaching (FLIP) method within a very narrow pH range 7.1-7.15. For CK-IAF without docked substrates, the time derivative of the initial loss of the fluorescent signal within the M-band equalled -3.26 at the fifth second and the decrease reached 82% by the 67th second. For CK-IAF with added substrates, the derivatives fell into the range of -0.95 to -1.30, with respective decreases from 16 to 46% at the 67th second. The results show that the substrates slowed down the exchange rate. This indicates that the strength of the bond between CK and the M-band of myofibrils increased.

On the basis of previous experience with biological effects of electromagnetic fields a potential effect of homogeneous sinusoidal magnetic field (50 Hz, 10 mT) on energy state of rat skeletal muscle was investigated. Two different total body exposures to magnetic field were selected: (1) repeated 1 hour exposure, 2 times a week for 3 months, and (2) acute 1.5 hour exposure (and the appropriate control groups). Important energy metabolites (adenosine triphosphate--ATP, creatine phosphate, creatine, lactate, pyruvate and inorganic phosphate) were analysed by enzymatic and spectroscopic methods in musculus gracilis cranialis. On the basis of the concentration of important energy metabolites the apparent Gibbs free energy of ATP hydrolysis and creatine charge was calculated. Our results demonstrate no influence of this low frequency magnetic field on the level of important energy metabolites in rat skeletal muscle. The conclusion of this study is that neither repeated exposure nor the acute exposure of rats to the sinusoidal magnetic field of given parameters has any important influence on the energy state of the skeletal muscle.

A method based on exposure of mice in the CAB 4.5 smoke chamber for the evaluation of the biological toxicity of the products of flame and smoldering combustion of polumeric materials has been developed. A toxicity index, characterizing the danger from the products of combustion of the test polymer under the given conditions, was proposed on the basis of values of the concentration of carboxyhaemoglobin in the blood of the test animals, attaining characteristic critical limits (lethal limit) and LC,,, values.

Human skeletal muscle metabolism is often investigated by measurements of substrate fluxes across the forearm. To evaluate whether the two forearms give the same metabolic information, nine healthy subjects were studied in the fasted state and during infusion of adrenaline. Both arms were catheterized in a cubital vein in the retrograde direction. A femoral artery was catheterized for blood sampling, and a femoral vein for infusion of adrenaline. Forearm blood flow was measured by venous occlusion strain-gauge plethysmography. Forearm subcutaneous adipose tissue blood flow was measured by the local 133Xe washout method. Metabolic fluxes were calculated as the product of forearm blood flow and a-v differences of metabolite concentrations. After baseline measurements, adrenaline was infused at a rate of 0.3 nmol kg-1 min-1. No difference in the metabolic information obtained in the fasting state could be demonstrated. During infusion of adrenaline, blood flow and lactate output increased significantly more in the non-dominant arm (8.12 +/- 1.24 versus 6.45 +/- 1.19 ml 100 g-1 min-1) and (2.99 +/- 0.60 versus 1.83 +/- 0.43 micromol 100 g-1 min-1). Adrenaline induced a significant increase in oxygen uptake in the non-dominant forearm (baseline period: 4.98 +/- 0.72 micromol 100 g-1 min-1; adrenaline period: 6.63 +/- 0.62 micromol 100 g-1 min-1) while there was no increase in the dominant forearm (baseline period: 5.69 +/- 1.03 micromol 100 g-1 min-1; adrenaline period: 4. 94 +/- 0.84 micromol 100 g-1 min-1). It is concluded that the two forearms do not respond equally to adrenaline stimulation. Thus, when comparing results from different studies, it is necessary to know which arm was examined.

In order to advance our understanding of the phenomenon of flow-induced increases in the metabolism of the relaxed muscle, the metabolic rate of the isolated rat gracilis muscle was investigated at 28 degrees C in vitro. The muscle was perfused with cell-free Krebs-Henseleit bicarbonate buffer containing 5% bovine serum albumin and 5 mM glucose, saturated with a gas mixture of 95% O2 and 5% CO2 and simultaneously superfused with a medium saturated with with a low O2 gas mixture (1% O2, 5% CO2 and 94% N2). Two different perfusion flow rates (0.054 and 0.100 ml min-1) have been used. Their influence on oxygen consumption and lactate production has been measured. After a 100 min perfusion period, the muscle was freeze-clamped and analysed for ATP, phosphocreatine, creatine, lactate, pyruvate, inorganic phosphate and glycogen content. The energy state of the cell and the proportions of glycolytic and mitochondrial fluxes of ATP synthesis were evaluated. During perfusion at the low flow rate of 0.054 ml min-1, the oxygen uptake was 45 +/- 9 nmol min-1 (g wet wt)-1, accompanied by a dominance of anaerobic glycolytic synthesis of ATP over mitochondrial ATP synthesis, even though the total delivery of oxygen to muscle was three times higher than oxygen consumption. Increasing the perfusion flow rate to 0.100 ml min-1 increased the oxygen uptake to 120 +/- 6 nmol min-1 (g wet wt)-1, thus leading to a prevalence of mitochondrial ATP synthesis over glycolytic ATP synthesis. The inner stores of glycogen served as the main substrate of energy metabolism and the role of exogenous substrates in the flow-stimulated increase of oxygen uptake was negligible. The increase in perfusion rate also enhanced the energy state of the muscle fibres, which was expressed either as the creatine charge or as the value of the change of Gibbs free energy of ATP hydrolysis. Data indicate that the change of perfusion flow rate per se, apart from oxygen and exogenous substrate supply, elicits changes in the regulation of energy metabolism within non-contracting skeletal muscle under open microcirculation.