Effects of the perfusion flow rate on skeletal muscle energy metabolism and a possible role of second messengers in this process.

The metabolic turnover in the isolated in vitro perfused and superfused rat skeletal muscle (musculus gracilis cranialis) was enhanced by increasing the medium flow rate under relaxed conditions. In a recent study we have measured the tissue concentrations of second messengers: cyclic adenosine 3'5'-monophosphate (cAMP), cyclic guanosine 3'5'-monophosphate (cGMP), and D-myo-inositol 1,4,5-trisphosphate (IP3) under similar experimental conditions to analyze their potential role in the described stimulation of metabolic rate by changes of perfusion flow rate. The tissue levels of the two second messengers' cAMP and cGMP were not significantly changed after increasing the perfusion flow rate and they probably have no transduction role in the induced alteration of skeletal muscle metabolism. However, the IP3 content was extremely reduced after increasing flow rate. This decrease in the tissue concentration of IP3 induced by increasing the flow rate indicates the possible role of IP3 in this signal transduction, leading to changes in the cellular metabolic pathways.

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 observed in the pH range tested (6.0-7.5). However, the same procedures resulted in a significant discrepancy between the amounts of reacted PCr and produced ATP within the pure myofibrillar fraction. This discrepancy represents the portion of ATP produced by the CK reaction, which is preferentially channelled to the myosin ATPase before diffusing into the bulk solution. The maximum evaluated difference reached 42.3 % at pH 6.95. The substrate channelling between myofibrillar-bound CK and myosin ATPase was evaluated under various pH levels within the physiological range and it reached a maximum value in a slightly acidic environment. These results suggest that ATP/ADP flux control by the CK system is more important at lower pH, corresponding to the physiological state of muscle fatigue.

Forearm metabolism during infusion of adrenaline: comparison of the dominant and non-dominant arm.

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.

Energy metabolism of rat skeletal muscle modulated by the rate of perfusion flow.

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.

Muscle ATP synthesis and utilisation, balanced during flow-induced increase of respiration.

To investigate the control of cell energetic metabolism, creatine charge, ATP/ADP ratio and oxygen consumption (as indicators of an energetic status, the balance between ATP synthesis and degradation and the aerobic ATP turnover, respectively) were evaluated in the rat gracilis muscle, perfused-superfused in vitro. During the perfusion rate of 70 microl/min the ATP/ADP ratio, as well as the creatine charge are kept at the in vivo level. With the decrease of the rate toward 54 microl/min (of an abundant oxygen delivery), the values of both parameters are lower than levels in vivo. With the increase of the rate up to 100 microl/min, both parameters are kept at the in vivo level, when respiration increases by 125%. The data demonstrate the 'unmatched' control of ATP utilisation and synthesis steady rates during the low perfusion rate; during the increasing steady ATP turnover following the increased perfusion rate, the two fluxes are strikingly 'matched', i.e. precisely balanced.

Thermogenic response to adrenaline during restricted blood flow in the forearm.

To elucidate the underlying mechanism behind the thermogenic effect of adrenaline in human skeletal muscle, nine healthy subjects were studied during intravenous infusion of adrenaline. Restriction of blood flow to one forearm was obtained by external compression of the brachial artery, to separate a direct metabolic effect of adrenaline from an effect dependent on increased blood flow. The other arm served as the control arm. In the control arm, the forearm blood flow increased 4.7-fold (from 2.0 +/- 0.3 to 9.3 +/- 1.5 mL 100 g(-1) min(-1), P < 0.001) during the adrenaline infusion. Adrenaline significantly increased forearm oxygen consumption (from 4.7 +/- 2.1 to 7.0 +/- 3.6 micromol 100 g(-1) min(-1), P < 0.025). In the arm with restricted blood flow, the forearm blood flow increased 2.9-fold (from 1.6 +/- 0.3 to 4.6 +/- 0.8 mL 100 g(-1) min(-1), P < 0.002) but the forearm oxygen consumption did not increase (baseline period: 5.6 +/- 2.3 micromol 100 g(-1) min(-1), adrenaline period: 6.1 +/- 2.1 micromol 100 g(-1) min(-1), P = 0.54). The experimental design and the difficulties in interpretation of the result are discussed. The results give evidence for the hypothesis that the vascular system plays a key role in the thermogenic effect of adrenaline in skeletal muscle in vivo.

Brown fat is essential for cold-induced thermogenesis but not for obesity resistance in aP2-Ucp mice.

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.

Levels of energy-related metabolites in intact and isolated perfused-superfused rat skeletal muscles.

Adenosine 5'-triphosphate (ATP), phosphocreatine (PCr), creatine (Cr), inorganic phosphate (Pi), lactate (LAC), pyruvate (PYR) and glycogen as glucose (GLU) were determined and free adenosine 5'-diphosphate (ADP) was calculated from ATP:creatine phosphokinase (CPK) reaction in the gracilis muscle of cold-acclimated rats in vivo, and in completely isolated muscles under medium perfusion and superfusion in vitro, using the freeze-clamping method. The mean in vivo levels (mumol/g w.w.) were: ATP 4.8, PCr 12.0, Cr 7.8, Pi 16.1, LAC 1.6, PYR 0.09, GLU 22.9, ADP 0.62 x 10(-3). Isolation of the muscle (about 11 min of anoxia followed by perfusion in the air with a high pO2 medium) decreased macroergic phosphate levels (ATP 3.0, PCr 8.3). In isolated muscles perfused with a high pO2 medium (99 kPa O2, perfusion rate 70 microliters/min) and simultaneously superfused with a low pO2 medium (6.2 kPa O2, 2.3 ml/min) at 28 degrees C in vitro the levels of metabolites were (mumol/g w.w.): ATP 3.1, PCr 8.5, Cr 5.6, Pi 9.2, LAC 2.1, PYR 0.19, GLU 6.6, ADP 0.44 x 10(-3). The mean steady oxygen uptake of the isolated muscle was 97 nmol O2 x min-1 x g-1 w.w. Thus, the levels of macroergic phosphates and their derivatives are lower after isolation and perfusion of the muscle, but the creatine charge [PCr]/([PCr]+[Cr]) remains stable (0.61 in vivo versus 0.60 in the isolated muscle). This indicates that the steady-state and high energy status of the isolated perfused-superfused gracilis muscle is maintained [corrected].

Quantitative aspects of the fever response elicited by intracerebral injection of endotoxin in the guinea-pig.

Fever developing after intracerebral injections of lipopolysaccharide (LPS) to guinea-pigs were monophasic, with only one peak of inner body temperature slowly developing and longlasting in a dose range 20 to 200 ng of LPS. Latency time was inversely related to the dose of LPS. Indomethacin injected to the third brain ventricle did not abolish fever response.

Static response in body temperature regulation of the euthermic warm-acclimated golden hamster (Mesocricetus auratus).

A characteristic feature of the body temperature regulation of euthermic golden hamsters is a great individual variability of body temperature in the thermoneutral zone. Resting values of the total metabolic rate (M) at ambient temperature 30-34 degrees C vary from 5.3 to 8.8 W.kg-1 between individuals, body temperature reaching 33.5-37.7 degrees C (subcutaneous temperature, Ts) and 35.4-39.0 degrees C (hypothalamic temperature, Th). The dependence of metabolic heat production on steady deviations of peripheral and central body temperature from the resting values in nonlinear in general, but the unknown functional relationship delta M = f (delta Th, delta Ts) can be replaced by a single linear regression function of Ts by neglecting the change of central body temperature: delta M = 2.14-2.00. delta Ts. Total body thermosensitivity of the golden hamster determined from steady changes of rectal temperature and metabolic rate after external cooling is -6.8 +/- 1.3 W.kg-1. degrees C-1.

Dynamic effects of acute cooling in body temperature regulation of the euthermic warm-acclimated golden hamster (Mesocricetus auratus).

During the dynamic phase of external cooling of euthermic golden hamsters in the initial period of metabolic response, peripheral body temperature is the decisive control variable determining the level of metabolic heat production. Under these conditions the rate as well as the magnitude of the peripheral body temperature change constitute the effectual input to the controller of body temperature. The apparent sensitivity with which the regulator drives the metabolic response to unit change of the peripheral temperature is in an inverse relation to the rate of peripheral temperature change. This parameter, despite its limited significance can serve as a working index characterising the thermoregulatory system in different groups of experimental animals of the same species providing that the actual conditions of the experiment are comporting.

The use of an intestinal thermode for studying thermoregulation of the golden hamster.

Intestinal cooling induces a normal metabolic response compensating heat loss in the euthermic golden hamster. The hypothalamic and subcutaneous temperatures change unpredictably and the threshold hypothalamic temperature for the induction of cold thermogenesis, similarly as the sensitivity of the regulator related to changes in hypothalamic temperature vary considerably, however. It seems that the thermal input from the hypothalamus does not contribute significantly to the control of cold thermogenesis in euthermic golden hamsters during intestinal cooling. The use of an intestinal thermode itself is not suitable for quantitative studies of thermoregulation in the golden hamster.