ConA induced changes in energy metabolism of rat thymocytes

The influence of ConA on the energy metabolism of quiescent rat thymocytes was investigated by measuring the effects of inhibitors of protein synthesis, proteolysis, RNA/DNA synthesis, Na⁺K⁺-ATPase, Ca²⁺-ATPase and mitochondrial ATP synthesis on respiration. Only about 50% of the coupled oxygen consumption of quiescent thymocytes could be assigned to specific processes using two different media. Under these conditions the oxygen is mainly used to drive mitochondrial proton leak and to provide ATP for protein synthesis and cation transport, whereas oxygen consumption to provide ATP for RNA/DNA synthesis and ATP-dependent proteolysis was not measurable. The mitogen ConA produced a persistent increase in oxygen consumption by about 30% within seconds. After stimulation more than 80% of respiration could be assigned to specific processes. The major oxygen consuming processes of ConA-stimulated thymocytes are mitochondrial proton leak, protein synthesis and Na⁺K⁺-ATPase with about 20% each of total oxygen consumption, while Ca²⁺-ATPase and RNA/DNA synthesis contribute about 10% each. Quiescent thymocytes resemble resting hepatocytes in that most of the oxygen consumption remains unexplained. In contrast, the pattern of energy metabolism in stimulated thymocytes is similar to that described for Ehrlich Ascites tumour cells and splenocytes, which may also be in an activated state. Most of the oxygen consumption is accounted for, so the unexplained process(es) in unstimulated cells shut(s) off on stimulation.     

Effects of methylprednisolone on the energy metabolism of quiescent and conA-stimulated thymocytes of the rat

The short-term effects of high concentrations of Methylprednisolone (MP) on the energy metabolism of quiescent and Concanavalin A-stimulated rat thymocytes were investigated in vitro. Concanavalin A (ConA) stimulated the respiration rate of quiescent thymocytes by 35%. Addition of more than 0.15 mg MP/10⁷ cells to ConA-stimulated cells reversed this respiratory stimulation; in addition, higher concentrations of MP caused a similar progressive decrease in the rate of respiration of both quiescent and ConA-stimulated cells. Similarly, the stimulation of respiration by ConA was greatly reduced in MP-treated cells. MP addition lowered cytoplasmic [Ca²⁺] and, at high concentrations, abolished the ability of ConA to increase [Ca²⁺]. Thus MP both reverses and prevents the immediate stimulation of thymocytes by ConA.In quiescent thymocytes, MP strongly inhibited that part of the oxygen consumption used to drive the cycle of Na⁺ influx across the plasma membrane and Na⁺ efflux on the Na⁺K⁺-ATPase, but did not inhibit oxygen consumption used to drive protein synthesis. In ConA-stimulated thymocytes MP had the same effects and also strongly inhibited oxygen consumption dependent on the cycle of Ca²⁺ influx across the plasma membrane and Ca²⁺ efflux on the Ca²⁺-ATPase, but had little effect on oxygen consumption used to drive RNA and DNA synthesis.These results show that MP prevents cation cycling in thymocytes (either by preventing cation influx or by inhibiting cation pumps) and prevents mitogenic stimulation of the cells. The high MP concentration required and the speed of onset of the effect (lless than 30s) provide strong evidence that these effects of MP are not mediated by glucocorticoid receptors and subsequent activation of gene expression. They may be caused by direct effects of MP on the properties of the plasma membrane. These effects are considered to be, at least partially, responsible for the beneficial results that currently have been obtained using MP megadoses in various clinical situations.     

Impairment of glucose metabolism and energy transfer in the hypertriglyceridemic rat heart

The metabolic pathways involved in ATP production in hypertriglyceridemic rat hearts were evaluated. Hearts from male Wistar rats with sugar-induced hypertriglyceridemia were perfused in an isolated organ system. Mechanical performance, oxygen uptake and beat rate were evaluated under perfusion with different oxidizable substrates. Age- and weight-matched animals were used as control. The hypertriglyceridemic (HTG) hearts showed a decrease in the mechanical work and slight diminution in the oxygen uptake when perfused with glucose, pyruvate or lactate. No differences were found when perfused with palmitate, octanoate or -hydroxybutyrate. The glycolytic flux in HTG hearts was 2.4 times lower than in control hearts. Phosphofructokinase-I (PFK-I) was 16% decreased in HTG hearts, whereas pyruvate kinase activity did not change. The increased levels of glucose-6hyphen;phosphate in HTG heart, suggested a flux limitation by the PFK-I. Pyruvate dehydrogenase in its active form (PDHa) diminished as well. The PDHa level in the HTG hearts was restored to control values by dichloroacetate; however, this addition did not significantly improve the mechanical performance. Levels of ATP and phosphocreatine as well as total creatine kinase activity and the MB fraction were significant lower in the HTG hearts perfused with glucose. The data suggested that supply of ATP by glucose oxidation did not suffice to support cardiac work in the HTG hearts; this impairment was exacerbated by the diminution of the creatine kinase system output.     

Effect of experimentally induced thyrotoxicosis on oxidative energy metabolism in rat heart mitochondria

Treatment of rats with T₃ resulted in a significant decrease in body weight, while the heart weight increased. T₄ treatment had less marked effect on body weights but resulted in decreased heart weights. Serum T₄ levels decreased significantly with simultaneous increase of T₃ level following T₃ treatment, whereas with T₄ treatment, levels of both T₄ and T₃ increased in the serum. Low doses of T₃ (0.5 μg ) caused decrease in mitochondrial protein content while high dose of T₄ (1 μg), caused significant increase in mitochondrial mass. The state 3 respiration rates were significantly depressed following T₃ and T₄ treatments, in a substrate specific manner with the effects being more pronounced with T₃; these responses with T₄ were dose-dependent for succinate and ascorbate + N,N,N′,N′-tetramethyl-p-phenylenedíamme. State 4 respiration rates also exhibited similar corresponding changes. ADP/O ratios were not changed but ADP-phosphorylation rates were decreased significantly particularly so with the T₃-treated animals. Treatment with T₃ also resulted in lowering of intramitochondrial cytochrome contents. Similar effects were seen also with higher doses of T₄. The results thus indicate that T_3- and T_4- thyrotoxicosis results in impaired energy metabolism in heart mitochondria.     

Circadian rhythm disorders and dynamic changes of energy metabolism in rat heart muscle cells

The functional states of pro- and antioxidant systems in blood and heart muscle cells in rats with long-term emotional stress have been studied. It has been shown that daily rhythm disorders produce psycho-emotional stress in animals and that, this is accompanied by quantitative changes in physiological parameters and hormones in the blood. In the present study, it was observed that such stress increased lipid peroxidation in blood and heart muscle cells. Also, activities of antioxidant enzymes, superoxide dismutase, and catalase were diminished, indicating deterioration of the antioxidant system. In addition, there were decreased activities of mitochondrial enzymes participating in energy metabolism, indicating decreased energy levels in heart muscle cells. These results suggest the likelihood that emotional stress is a key factor that can cause a whole range of diseases of the cardiovascular system.     

Temperature-dependent changes in energy metabolism, intracellular pH and blood oxygen tension in the Atlantic cod

The effect of acute increase in temperature on oxygen partial pressure (Po ₂) was measured in the gill arches of Atlantic cod Gadus morhua between 10 and 19° C by use of oxygen microoptodes. Oxygen saturation of the gill blood under control conditions varied between 90 and 15% reflecting a variable percentage of arterial or venous blood in accordance with the position of each optode in the gill arch. The data obtained suggested that arterial Po₂ remained more or less constant and arterial oxygen uptake did not become limiting during warming. A progressive drop in venous Po₂, however, was observed at >10° C indicating that excessive oxygen uptake from the blood is not fully compensated for by circulatory performance, until finally, Po₂ levels fully collapse. In a second set of experiments energy and acid–base status of white muscle of Atlantic cod in vivo was measured by magnetic resonance (³¹P‐NMR) spectroscopy in unanaesthetized and unimmobilized fish in the temperature range between 13 and 21° C. A decrease in white muscle intracellular pH (pH_i) with temperature occurred between 10 and 16° C (ΔpH per ° C = ?0·025 per ° C). In white muscle temperature changes had no influence on high‐energy phosphates such as phosphocreatine (PCr) or ATP except during exposure to high critical temperatures (>16° C), indicating that white muscle energy status appears to be relatively insensitive to thermal stress if compared to the thermal sensitivity of the whole animal. The data were consistent with the hypothesis of an oxygen limitation of thermal tolerance in animals, which is set by limited capacity of oxygen supply mechanisms. In the case of Atlantic cod circulatory rather than ventilatory performance may be the first process to cause oxygen deficiency during heat stress.     

The effect of acclimation temperature on energy metabolism as a function of body weight in rats

1. 1|Energy metabolism of 38 rats acclimated to 10, 20 and 30°C was measured at these ambient temperatures and the regression equations over body weight were calculated.

2. 2|Expressing metabolic rate as M = kWⁿ, the results showed that the value of n is apparently variable according to the acclimation and measurement temperatures.

3. 3|It was also shown that the 2/3 power of body weight is a suitable parameter to express metabolic rate, regardless of acclimation or environmental temperatures.

Changes of synaptosomal energy metabolism induced by hypoxia during aging

Synaptosomes were isolated from the motor area of the cerebral cortex of normoxic or hypoxic (PaO₂=17–19 mmHg, for 15 min) beagle dogs of different ages. Synaptosomes were incubated in Krebs-Henseleit-Hepes buffer (for 10 min at 24°C) and the energetic state was defined by: the balance of the labile phosphates (ATP, ADP, AMP, and creatine phosphate); the respiratory rate; the redox state of the intramitochondrial NAD-couple. By the present experimental model, it is possible to evaluate the potential damage (induced by the in vivo hypoxic insult) that synaptosomes cannot reverse under optimal incubation. Aging affected the phosphorylation state of the post-hypoxic incubated synaptosomes. The oxygen consumption rate was quite similar in the synaptosomal fractions from the motor area of hypoxic beagle dogs of different ages, but the cytochromec anda contents were lower in the preparations from hypoxic older brains. In dogs of different ages, hypoxia always lowered the respiration of the synaptosomes, but aging affected the oxygen consumption rates only in post-hypoxic synaptosomes incubated with succinate. The synaptosomal energetic state was defined also by the redox state of the intramitochondrial NAD-couple (G_ox-red) and the phosphorylation state of adenine nucleotide system (G_ATP). The free-energy change (G) for the coupled reactions was calculated, too. In synaptosomes isolated from the cerebral cortex of dogs submitted to hypoxia, the equilibrium (calculated for the mitochondrial electron transfer chain and the phosphorylation of adenine nucleotides) was markedly altered as function of aging. The extensive age-related G changes were largely supported by alteration of the phosphorylation state of adenine nucleotides, rather than by modification of the redox state of the electron transfer chain.All present data suggest that the bioenergetic derangement caused by hypoxia and aging may be interpreted also in terms of modification of the biophysical and biochemical mechanisms involving the mitochondrial membranes and particularly the inner mitochondrial membrane.Special Issue Dedicated to Dr. Abel Lajtha.     

Effect of a ubiquinone-like molecule on oxidative energy metabolism in rat cortical synaptosomes at different ages

Persistent stimulation of energy consumption, induced by depolarization with veratridine, mimics a condition of abnormally enhanced energy demand and causes an increase in the oxygen consumption rate (QO₂) and in the interconversion of pyruvate dehydrogenase complex (PDHc) into its active form. Wistar rats at the age of 26 months do not show alterations of QO₂ and of the ability of veratridine to increase QO₂ in comparison with 6 month-old animals whereas the active form of PDHc is slightly but significantly reduced. Idebenone, a ubiquinone-like molecule (1 M), does not affect the QO₂ or PDHc activation state in resting conditions but attenuates the veratridine-challenged increase in QO₂ at all the ages tested and attenuates the increase in the percentage of PDHa reaching statistical significance in 26-month-old rats. At higher concentration (10 M) idebenone totally abolishes the veratridine-induced increase in PDHa also in the 6 month-old rats. At the lower concentration, the drug does not affect the increase in QO₂ induced by an uncoupler of oxidative phosphorylation. The results obtained suggest a protective effect of idebenone on the cerebral tissue against stressful conditions; this action may be exerted at the level of some mitochondrial component and/or on the Na⁺ homeostasis.     

The changes in the energy metabolism of human muscle induced by training

The biochemical effects of training programmes have been studied with a kinetic model of central metabolism, using enzyme activities and metabolite concentrations measured at rest and after 30 s maximum-intensity exercise, collected before and after long and short periods of training, which differed only by the duration of the rest intervals. After short periods of training the glycolytic flux at rest was three times higher than it had been before training, whereas during exercise the flux and energy consumption remained the same as before training. Long periods of training had less effect on the glycolytic flux at rest, but increased it in response to exercise, increasing the contribution of oxidative phosphorylation.     

中华倒刺鲃能量代谢和热耐受特征的体重效应

为了探讨体重对中华倒刺鲃能量代谢和热耐受特征的影响,在25℃条件下分别测定不同体重大小(1、10、25、>50 g组)中华倒刺鲃的静止代谢率(Resting metabolic rate,RMR)、力竭运动后过量耗氧(Excess post-exercise oxygen consumption,EPOC)和热耐受参数。随体重的增加,中华倒刺鲃个体RMR、最大代谢率(Maximum metabolic rate,MMR)、代谢空间(Metabolic scope,MS)和EPOC均显著增加(P<0.05);单位体重RMR、MMR和MS则均显著降低,而单位体重EPOC随体重的增加显著增加(P<0.05)。RMR、MMR、MS和EPOC的代谢尺度指数分别为0.796、0.834、0.849和1.137且显著大于2/3,因此中华倒刺鲃的能量代谢参数均呈异速度增加的关系而变化。这些变化可能与其个体变大游泳运动能力增强导致能量代谢需求增加相关。中华倒刺鲃临界低温(Critical thermal minimum,CT_min)和致死低温(Lethal the...     

利用核磁共振同时观测大鼠心室内压参数与能量代谢

本文介绍一种在核磁共振（ｎｕｃｌｅａｒ ｍａｇｎｅｔｉｃ ｒｅｓｏｎａｎｃｅ,ＮＭＲ）谱仪上对大鼠主室内压参数与能量代谢同时测定的技术。该方法采用离体等容大鼠心功能监测系统测量和分析心脏的心室内压参数,通过测定心脏的磷－３１核磁共振（＾３１Ｐ ＮＭＲ）谱观测心肌组织的能量代谢状态。     

中缅树鼩能量代谢的季节变化

小型哺乳动物的体重和产热特征的季节调节对其生存至关重要。为探讨中缅树鼩的能量代谢适应特征随季节的变化,采用耗氧量测定、食物平衡法、形态测量等方法,分别对其冬季和夏季的基础代谢率(BMR)、非颤抖性产热(NST)、体温、体重、蒸发失水、能量收支和消化道的长度和重量进行了测定。中缅树鼩冬季体温、体重、基础代谢率、NST、蒸发失水散热分别为37. 9℃ ± 0.14℃ ,126.1 ± 2.1 g,42. 94 ± 2.65 J/g· h,54. 97 ±2.14 J/ g·h,5. 69 ±0.33 J/ g·h;夏季体温、体重、基础代谢率、NST、蒸发失水散热分别为38.5℃ ± 0. 27℃ ,106.9 ±5.1 g,28. 69 ±3.06 J/ g·h,47.43 ± 2.45 J / g·h,7.12 ±0. 57 J/ g·h;中缅树鼩的每日摄入能、消化能、可代谢能冬季均比夏季显著增加,消化道特征冬季和夏季存在变化,随着温度降低、食物质量下降,小肠长度和重量增加。这些结果表明:中缅树鼩在冬季,通过增加体重、基础代谢率和NST、能量摄入、消化能和可代谢能,降低蒸发失水等方式应对季节性环境变化。代谢产热和消化生理调节在季节性适应过程中具有重要地位。     

Interaction of the B subunit of cholera toxin with endogenous ganglioside GM1 causes changes in membrane potential of rat thymocytes

Summary The fluorescent anionic dye, bisoxonol, and flow cytometry have been used to monitor changes in the membrane potential of rat thymocytes exposed to the B subunit of cholera toxin. The B subunit induced a rapid hyperpolarization, which was due to activation of a Ca²⁺-sensitive K⁺ channel. Reduction of extracellular Ca²⁺ to <1 m by the addition of [ethylenebis(oxyethylenenitrilo)]tetraacetic acid immediately abolished the hyperpolarization caused by the B subunit. Cells treated with quinine and tetraethylammonium lost their ability to respond to the B subunit, whereas 4-aminopyridine did not have any effect. Thus, calcium-sensitive and not voltage-gated K⁺ channels appeared to be responsible for the hyperpolarization. The results of ion substitution experiments indicated that extracellular Na⁺ was not essential for changes in membrane potential. Further studies with ouabain, amiloride and furosemide demonstrated that electrogenic Na⁺/K⁺ ATPase, Na⁺/H⁺ antiporter and Na⁺/K⁺/Cl^– cotransporter, respectively, were not involved in the hyperpolarization process induced by the B subunit. Thus, crosslinking of several molecules of ganglioside GM1 on the cell surface of rat thymocytes by the pentavalent B subunit of cholera toxin modulated plasma membrane permeability to K⁺ by triggering the opening of Ca²⁺-sensitive K⁺ channels. A role for gangliosides in regulating ion permeability would have important implications for the function of gangliosides in various cellular phenomena.     

Plasminogen activator and protease inhibitor activities in isolated rat thymocytes

Summary Plasminogen Activator (PA) and its response to glucocorticoids and androgens was studied in viable rat thymocytes in suspension. PA was measured by its ability to convert plasminogen to plasmin, and the formed plasmin determined by cleavage of ¹⁴C-labeled globin. Using this functional assay, PA was found to be associated with the outer surface of thymic cells, and only negligible activity recovered from the incubation medium. Rat thymocytes also contain cytoplasmic and nuclear inhibitor(s) of the serine proteases plasmin, trypsin, chymotrypsin and thymic PA. Release of these inhibitors prevented determination of thymic PA activity in presence of lysed cells.The specific activity of PA in thymocytes isolated from adrenalectomized-castrated rats did not differ significantly from the specific activity associated with cells from intact animals. Furthermore, treatment of adrenalectomized-castrated rats with 0.1 mg of dexamethasone/ kg for 2 days induced thymic involution without affecting thymic PA activity. These observations suggest that PA activity of thymocytes is not involved in glucocorticoid-mediated thymic involution.     

Clusterin mRNA expression in apoptotic and activated rat thymocytes

Clusterin is a 75-80 kDa heterodimeric glycoprotein,that is produced in most tissues but which exact biological role is still not clear.Pwarticularly,its role in protection or promotion of apoptosis is heavily disputed,since data supporting both views have been reported in several independent sutdies.To clarify this issue,and also to determine whether clusterin expression itself might be affected by apoptosis,in the present study,rat thymocytes were treated with dexamethasone,-a synthetic glucocorticoid that elicits apoptosis in thymocytes-,and clusterin mRNA expression was analyzed by semi-quantitative RT-PCR before and after induction of apoptosis.Interestingly,neither the treatment with dexamethasone in vitro nor triggering of apoptosis in vivo up-regulated clusterin expression,opposing the view that clusterin is involved in apoptotic processes.On the other hand,a new clusterin mRNA isoform was detected and isolated,whose expression ws restricted to freshly isolated thymocytes.This novel isoform lacks the post-translational proteolytic cleavage site and is therefore predicted to encode a monomeric protein.The biological function under normal circumstances,however,will nedd further investigations for clarification.While apoptosis could not modulate clusterin expression,activation of thymocytes with concanavalin A and interleukin-2 resulted in up-regulation of clusterin mRNA level,indicating that clusterin expression is rather under the control of cell activation-mediated rather than apoptosis-induced signals.     

Regional cerebral energy metabolism in bicuculline induced seizures

In order to assess the early regional changes in energy metabolism in bicuculline induced seizures, mice were injected and sacrificed before the onset of overt seizure activity, and shortly after clonic-tonic seizures began. The energy metabolites glucose, ATP, and phosphocreatine were measured in layers of the motor cortex and the cerebellar vermis. Results showed minimal metabolite changes in the cerebellum, whereas changes in energy metabolism in the motor cortex were largely localized to the layers containing pyramidal cells. These results are in agreement with previous studies showing a relative sparing of the cerebellum, and suggest early cortical changes occur in pyramidal cells.     

Pentylenetetrazole induced changes in cerebellar energy metabolism

Pentylenetetrazole was administered to Swiss-Albino mice, producing clonic-tonic seizures. Other groups were pretreated with one of the three anticonvulsants: phenytoin, clonazepam, or sodium valproate. Mice were sacrificed during the preseizure (1 minute) stage and at the onset of clonic-tonic seizures (2 minutes). Glucose, glycogen, ATP, and phosphocreatine were measured in layers of the parietal cortex and cerebellar vermis. Cortical metabolites were unchanged, or increased slightly, suggesting decreased utilization. In both cerebellar layers, glucose and glycogen were significantly decreased, and phosphocreatine was decreased in the molecular layer. These results indicate a regionally selective effect for pentylenetetrazole on cerebral energy metabolites. Pretreatment with anticonvulsants reduced the severity of the seizure, and eliminated the effect of pentylenetetrazole on glucose and glycogen.