Objective-Besides its well-described metabolic effects, vascular AMP-activated protein kinase (AMPK) can activate endothelial NO synthase, promotes angiogenesis, and limits endothelial cell apoptosis. The current study was designed to study the effects of alpha 1AMPK deletion during vascular disease in vivo.Methods and Results-Chronic angiotensin II infusion at low subpressor doses caused a mild endothelial dysfunction that was significantly aggravated in alpha 1AMPK-knockout mice. Unexpectedly, this endothelial dysfunction was not associated with decreased NO content, because NO levels measured by serum nitrite or electron paramagnetic resonance were even increased. However, because of parallel superoxide production, NO was consumed under production of peroxynitrite in angiotensin II-treated alpha 1AMPK-knockout mice, associated with NADPH oxidase activation and Nox2 upregulation. As Nox2 is also a component of phagocyte NADPH oxidases, we found a vascular upregulation of several proinflammatory markers, including inducible NO synthase, vascular cell adhesion molecule-1, and cyclooxygenase-2. Cotreatment with the NADPH oxidase inhibitor apocynin was able to prevent vascular inflammation and also partially restored endothelial function in alpha 1AMPK-knockout mice.Conclusion-Our data indicate that in vivo alpha 1AMPK deletion leads to Nox2 upregulation, resulting in endothelial dysfunction and vascular inflammation. This implicates basal AMPK activity as a protective, redox-regulating element in vascular homeostasis. (Arterioscler Thromb Vasc Biol. 2011;31:560-566.)

SUMMARY Trehalase (EC 3.2.1.28) hydrolyzes the main haemolymph sugar of insects, trehalose, into the essential cellular substrate glucose. Trehalase in locust flight muscle is bound to membranes that appear in the microsomal fraction upon tissue fractionation, but the exact location in vivo has remained elusive. Trehalase has been proposed to be regulated by a novel type of activity control that is based on the reversible transformation of a latent (inactive) form into an overt (active) form. Most trehalase activity from saline-injected controls was membrane-bound (95%) and comprised an overt form (∼25%) and a latent form (75%). Latent trehalase could be assayed only after the integrity of membranes had been destroyed. Trehazolin, a potent tight-binding inhibitor of trehalase, is confined to the extracellular space and has been used as a tool to gather information on the relationship between latent and overt trehalase. Trehazolin was injected into the haemolymph of locusts, and the trehalase activity of the flight muscle was determined at different times over a 30-day period. Total trehalase activity in locust flight muscle was markedly inhibited during the first half of the interval, but reappeared during the second half. Inhibition of the overt form preceded inhibition of the latent form, and the time course suggested a reversible precursor–product relation (cycling) between the two forms. The results support the working hypothesis that trehalase functions as an ectoenzyme, the activity of which is regulated by reversible transformation of latent into overt trehalase.

Trehalose is the main haemolymph sugar in many insect species. To be utilized trehalose must be hydrolysed into its glucose units by trehalase (EC 3.2.1.28). Inhibitors of trehalase have attracted interest as possible pesticides and tools for studying the regulation of trehalose metabolism in insects. To make full use of these inhibitors requires knowledge of their fate and effects in vivo. To this end we have measured trehazolin in locusts using a method based on the specific inhibition of a trehalase preparation. After injection of 20 μg, trehazolin decreased in haemolymph with a half-life of 2.6 days and after 10 days almost 95% had disappeared. Trehazolin did not reach the intracellular water space of locust tissues, but appeared with full inhibitory potency in locust faeces, suggesting that it was not metabolized, but quantitatively eliminated via the gut. Haemolymph trehalose increased transiently upon trehazolin injection, it was maximal after 3 days, then decreased and reached control level after 10 days. Inhibition of flight muscle trehalase by trehazolin was prolonged and still conspicuous 21 days post injection, suggesting that trehazolin inhibits trehalase activity irreversibly in vivo and that recovery requires de novo enzyme synthesis.

Previously, the cDNA and the respective gene for a presumed tauropine dehydrogenase (TaDH) from Suberites domuncula (GenBank accession nos. AM712888, AM712889) had been annotated. The conclusion that the sequences encode a TaDH had been inferred from the 68% identity with the TaDH protein from the marine demosponge Halichondria japonica. However, subsequent enzymatic assays shown here indicate that the presumed S. domuncula opine dehydrogenase is in fact a strombine dehydrogenase (StDH). The enzyme StDH is highly specific for glycine and is inhibited by an excess of the substrate pyruvate. Besides kinetic data, we report in this study also on the predicted tertiary and quaternary structure of the sponge StDH. It is concluded that the dimer (75 kDa) has a novel structure, distinguishing it from other known marine invertebrate OpDHs that exist as monomers.

Standard ecological methods (pitfall traps, trunk eclectors and soil cores) were used to evaluate collembolan community responses to different flooding intensities. Three sites of a floodplain habitat near Mainz, Germany, with different flooding regimes were investigated. The structures of collembolan communities are markedly different depending on flooding intensity. Sites more affected by flooding are dominated by hygrophilic and hygrotolerant species, whereas the hardwood floodplain is dominated by mesophilic species. The survival strategies of the hygrophilic and hygrotolerant species include egg diapause and passive drifting. The physiological adaptations to hypoxic conditions of several collembolan species were analyzed using a microcalorimeter. The activities were tested under normoxic and hypoxic/anoxic conditions as well as during post-hypoxic recovery. Lactate was increased after hypoxic intervals in the species studied, suggesting that, in addition to a massive decrease in metabolic rate, a modest glycolytic activity may be involved in the tolerance to hypoxia.

Boar spermatozoa contain isoforms of both glyceraldehyde 3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) and pyruvate kinase (PK, EC 2.7.1.40). The sperm-specific forms, GAPDH-S and PK-S, are tightly bound to cell structures. By immunofluorescence microscopy GAPDH-S and PK-S were localised in the principal piece of the boar sperm flagellum as well as in the acrosomal region of the sperm head and at the head-midpiece junction. The midpiece of the flagellum, however, contains isoforms of GAPDH and PK that were only recognised by antibodies against somatic GAPDH and PK, respectively, but not by the antibodies against GAPDH-S and PK-S. In sections of boar testis, GAPDH-S and PK-S were first detected in elongating spermatids when both the developing flagellum and the head were labelled with antibodies against GAPDH-S and PK-S. In contrast, antibodies against rabbit muscle GAPDH and PK labelled all developmental stages of germ cells and also neighbouring contractile cells. Thus, the structure-bound sperm-specific enzymes, GAPDH-S and PK-S, appeared only late in spermatogenesis simultaneously with the development of the structures to which they are bound. Anchoring glycolytic enzymes to structures in these mitochondria-free regions may secure ATP-production for both motility and acrosome function.

Boar spermatozoa contain a novel pyruvate kinase (PK-S) that is tightly bound at the acrosome of the sperm head and at the fibrous sheath in the principal piece of the flagellum, while the midpiece contains a soluble pyruvate kinase (PK). PK-S could not be solubilized by detergents, but by trypsin with no loss of activity. Purified PK-S as well as PK-S still bound to cell structures and soluble sperm PK have all kinetics similar to those of rabbit muscle PK-M1. The PK-S subunit had a relative molecular mass of 64 +/- 1 x 10(3) (n = 3), i.e. slightly higher than that of PK-M1, and carried an N-terminal extension (NH(2)-TSEAM-COOH) that is lacking in native PK-M1. Evidence is provided that PK-S is encoded by the PKM gene. Antibodies produced against the N-terminus of purified PK-S (NH(2)-TSEAMPKAHMDAG-COOH) were specific for PK-S as they did not react with somatic PKs or soluble sperm PK, while anti-PK-M1 recognized both sperm PKs. Immunofluorescence microscopy showed anti-PK-S to label the acrosome and the flagellar principal piece, whereas the midpiece containing the mitochondria was labelled only by anti-PK-M1. Immunogold labelling confirmed the localization of PK-S at the acrosome. In the principal piece, both polyclonal anti-PK-M1 and anti-PK-S were found at the fibrous sheath. Our results suggest that PK-S is a major component in the structural organization of glycolysis in boar spermatozoa.

The wasp Ampulex compressa injects a cocktail of neurotoxins into the brain of its cockroach prey to induce an enduring change in the execution of locomotory behaviors. Our hypothesis is that the venom injected into the brain indirectly alters the activity of monoaminergic neurons, thus changing the levels of monoamines that tune the central synapses of locomotory circuits. The purpose of the present investigation was to establish whether the venom alters the descending control, from the brain, of octopaminergic neurons in the thorax. This question was approached by recording the activity of specific identified octopaminergic neurons after removing the input from the brain or after a wasp sting into the brain. We show that the activity of these neurons is altered in stung and "brainless" animals. The spontaneous firing rate of these neurons in stung and brainless animals is approximately 20% that in control animals. Furthermore, we show that an identified octopamine neuron responds more weakly both to sensory stimuli and to direct injection of current in all treated groups. The alteration in the activity of octopamine neurons is likely to be part of the mechanism by which the wasp induces a change in the behavioral state of its prey and also affects its metabolism by reducing the potent glycolytic activator fructose 2,6-bisphosphate in leg muscle. To our knowledge, this is the first direct evidence of a change in electrical activity of specific monoaminergic neurons that can be so closely associated with a venom-induced change in behavioral state of a prey animal.

Insect flight is one of the most intense and energy-demanding physiological activities. High carbohydrate oxidation rates are necessary for take-off, but, to spare the limited carbohydrate reserves, long-distance flyers, such as locusts, soon switch to lipid as the main fuel. We demonstrate that before a flight, locust muscles are metabolically poised for take-off by the release of octopamine from central modulatory dorsal unpaired median (DUM) neurons, which increases the levels of the potent glycolytic activator fructose 2,6-bisphosphate in flight muscle. Because DUM neurons innervating the flight muscles are active during rest but selectively inhibited during flight, they stimulate carbohydrate catabolism during take-off but tend to decrease muscle glycolysis during prolonged flight. cAMP-dependent protein kinase A is necessary but not sufficient for signal transduction, suggesting parallel control via a calcium-dependent pathway. Locust flight is the first reported instance of a direct and specific involvement of neuronal activity in the control of muscle glycolysis in working muscle during exercise.

Endothelial cells line the intimal surface of blood vessels forming the interface between blood and tissue. Endothelial cells are unique in that they can form new capillaries from preexisting blood vessels (angiogenesis). Angiogenesis in vivo is a complex and highly regulated process, which becomes dysregulated under pathological conditions such as tumor growth, diabetic retinopathy and psoriasis. The fundamental significance of angiogenesis in these diseases has resulted in extensive research for pharmaceuticals affecting angiogenesis. We have established an in vitro model of angiogenesis which utilizes human endothelial cells (Figure 1) and have defined criteria for a software-supported image quantification (SSIQ) for analyzing the effects of compounds on angiogenesis.

Hypertrehalosaemic hormones stimulate trehalogenesis while inhibiting glycolysis in cockroach fat body. Signal transduction of the hypertrehalosaemic peptide Bld HrTH was examined in isolated fat body of the Argentine cockroach Blaptica dubia with respect to its effects on the increase in trehalose production and decrease in the content of the glycolytic activator fructose 2,6-bisphosphate in the tissue. Cyclic AMP does not seem to be involved in these processes as the cAMP analogue cpt-cAMP and the phosphodiesterase inhibitor IBMX, which both permeate cell membranes, had no effect on either parameter. Octopamine at physiological concentrations (10−7 mol · l−1) was also ineffective, but at 10−5 mol · l−1 or above, octopamine stimulated trehalose production although the content of fructose 2,6-bisphosphate in fat body was not affected. Both calcium entry and the release of Ca2+ from intracellular stores seem to be involved in the action of the hormone. If Ca2+ was omitted from the incubation medium, the hormone stimulated trehalose production less, though still significantly, whereas the hormone effect on fructose 2,6-bisphosphate was completely abolished in the absence of extracellular Ca2+. With Ca2+ present in the medium, the effect of the hormone on fructose 2,6-bisphosphate could be fully mimicked by the calcium ionophore A23187, suggesting that calcium entry is a␣decisive step in this signalling pathway. Trehalose production, on the other hand, was increased by thimerosal and thapsigargin which increase cytosolic Ca2+ from intracellular stores, whereas thimerosal in the absence of extracellular Ca2+ increased rather than decreased the content of fructose 2,6-bisphosphate, thus dissociating the two effects, which are normally coordinated by the hormone. Trehalose production and the content of fructose 2,6-bisphosphate were not significantly affected by mepacrine and mellitin, which are known to inhibit, respectively stimulate, phospholipase A2. Our data suggest that the effects of Bld HrTH on the stimulation of trehalose production and reduction of fructose 2,6-bisphosphate content in fat body are mediated by Ca2+, but that different signalling pathways are involved, suggesting that the two processes, although they are functionally linked, could be regulated separately.

The aim of this study was to assess the acute, sublethal effects of chemicals on fish from freshwater habitats. To this end goldfish (Carassius auratus) were exposed for up to 48 h to 2,4-dinitrophenol at concentrations well below the LC50 (96 h). In some experiments, fish were exposed to 2,4-dinitrophenol plus hypoxia/anoxia as an additional metabolic stress in order to enhance the effects of the chemical. Microcalorimetry proved very sensitive for detecting metabolic changes induced by 2,4-dinitrophenol. At 6 mg · 1−1 (corresponding to 14 LC50) the chemical had no significant effects on behaviour, motor activity and ventilation frequency, whereas the heat flow rate was markedly increased. Zebrafish Brachydanio rerio were unable to tolerate severe hypoxia, whereas goldfish proved very tolerant of hypoxia/anoxia. In goldfish, hypoxia induced a marked decrease in heat production, to less than 30% of the normoxic rate. Postanoxic recovery after 3–7 h of anoxia was rapid and complete; it was characterised by a transient period of excess heat. 2,4-Dinitrophenol affected the rate of aerobic heat production. It had no significant effect during hypoxia/anoxia but it markedly increased the heat flow rate during postanoxic recovery. As an adaptation to anoxia, goldfish produce and excrete ethanol during anaerobiosis. This process was not affected by 2,4-dinitrophenol. Microcalorimetry appears to be a sensitive method to detect metabolic effects of environmental chemicals.

Microcalorimetry was used to study the effects of graded hypoxia and anoxia on two species of insects that differ in their tolerance of anoxia. Locusts (Locusta migratoria) can survive an atmosphere of pure nitrogen for not more than 4 h (at room temperature), whereas hawk moths (Manduca sexta) can recover from more than 24 h of anoxia.To produce graded hypoxia, air and pure nitrogen were mixed and this mixture was passed through the cells of a twin calorimeter equipped with circulation cells. A gas flow containing 2% or more of oxygen had no significant effect on behaviour (as observed in parallel experiments using transparent cells) or heat flow rate. If oxygen content was reduced to 1% or less the effects of oxygen lack became conspicuous; at a ''critical oxygen concentration'' (between 2 and 1% oxygen) the animals became agitated; they hyperventilated and showed escape movements, which were followed by a loss of body posture and complete immobility within a few minutes.This behaviour was reflected by a distinct peak in heat flow rate followed by a precipitous decrease in heat flow below the normoxic rate. During graded hypoxia, the heat flow rate approached a new value which was correlated with the degree of hypoxia. Under strict anoxia, 5.3 +/- 1.1% and 3.6 +/- 1.8% of the normoxic heat flow rates were reached by Locusta and Manduca, respectively. Thus the two insect species reacted similarly with respect to behaviour and metabolic rate (as indicated by heat flow) to both graded hypoxia and anoxia. A striking difference between the species was seen, however, in the rate of heat flow during recovery from hypoxia or anoxia. Readmittance of air after an anoxic or hypoxic interval led to a rapid increase in heat flow above the normoxic rate, but the amount of ''excess heat production'' during recovery was much higher in Locusta than in Manduca. The difference in the amount of ''excess heat production'' during posthypoxic recovery could not be accounted for by differences in behaviour (muscular activity) or by the effect of ambient temperature.

A perfused locust thoracic muscle preparation was used to study the effects of octopamine on the content of fructose 2,6-bisphosphate, a potent activator of the glycolytic key enzyme phosphofructokinase, in the flight muscle of the locust Locusta migratoria. Perfusion with octopamine resulted in a significant increase in fructose 2,6-bisphosphate in flight muscle. The naturally occuring d-isomer was more potent than both d,l-octopamine and l-octopamine and gave a significant effect at 10−7 M, a concentration obeserved in the haemolymph of flying locusts. The adipokinetic hormones AKH I and AKH II of Locusta had no effect on fructose 2,6-bisphosphate in perfused flight muscle. Taurine which can be found in high concentrations in locust flight muscle was also ineffective. Electrical stimulation of perfused flight muscle (at 2 or 4 Hz for 15 min) caused the content of fructose 2,6-bisphosphate in flight muscle to decrease. Electrical stimulation, however, could not reverse the effect of octopamine added to the perfusion medium. It is suggested that octopamine-stimulated elevation of fructose 2,6-bisphosphate contributes to activation of glycolysis during locust flight.

In the flight muscle of resting locusts the ratio of phosphoarginine to ATP was the same whether determined by NMR (1.76) or biochemically, but the NMR-visible content of inorganic phosphate (Pi) was only 40% of ATP, i.e., much lower than total Pi as determined biochemically. This suggests that most of the Pi in flight muscle is not free, and hence not available as substrate or effector for cytosolic enzymes. Similarly, the free content of ADP and AMP in resting muscle was calculated to be much lower than the total content.

Heat production of male frogs, Rana temporaria, was measured in a microcalorimeter through which a continuous flow of gas was passed in order to generate constant normoxic, hypoxic or anoxic conditions. The normoxic heat flow was 163 ± 37 μ Wg body weight in frogs that had not been treated with curare and 149 ± 69 μ Wg in animals immobilized with curare. During anoxia, frogs, whether curarized or not, decreased their heat production to about 25% of the respective normoxic control. In graded hypoxia (10% to 3% O2), curarized frogs decreased their heat rate according to the grade of hypoxia they were subjected to.

Conference Article| May 01 1992 2-AMINOETHYLPHOSPHONIC ACID IS THE MAIN PHOSPHORUS COMPOUND IN LOCUST HAEMOLYMPH PETER M. KILBY; PETER M. KILBY * 1Department of Biochemistry, University of Oxford, South Parks Road, OX1 3QU Search for other works by this author on: This Site PubMed Google Scholar GERHARD WEGENER; GERHARD WEGENER 2Institut für Zoologie, Johannes Gutenberg-Universität, Saarstrasse 21, D6500 Mainz, Germany Search for other works by this author on: This Site PubMed Google Scholar GEORGE K. RADDA GEORGE K. RADDA 1Department of Biochemistry, University of Oxford, South Parks Road, OX1 3QU Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1992) 20 (2): 220S. https://doi.org/10.1042/bst020220s Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Twitter LinkedIn Cite Icon Cite Get Permissions Citation PETER M. KILBY, GERHARD WEGENER, GEORGE K. RADDA; 2-AMINOETHYLPHOSPHONIC ACID IS THE MAIN PHOSPHORUS COMPOUND IN LOCUST HAEMOLYMPH. Biochem Soc Trans 1 May 1992; 20 (2): 220S. doi: https://doi.org/10.1042/bst020220s Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search This content is only available as a PDF. © 1992 Biochemical Society1992 Article PDF first page preview Close Modal You do not currently have access to this content.

White skeletal muscle of crucian carp contains a single isoenzyme of glycogen phosphorylase, which was purified approximately 300-fold to a specific activity of approximately 13 mumol.min-1.mg protein-1 (assayed in the direction of glycogen breakdown at 25 degrees C). Tissue extracts of crucian muscle produced three distinct peaks of phosphorylase activity when separated on DEAE-Sephacel. Peaks 1 and 3 were identified, in terms of kinetic properties and by interconversion experiments, as phosphorylase b and a, respectively. Peak 2 was shown to be a phospho-dephospho hybrid. The three interconvertible forms of phosphorylase were purified and shown to be dimeric molecules at 20 degrees C. At 5 degrees C, a and the hybrid tended to form tetramers. The Mr of the subunit was estimated to be 96,400 from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The hybrid is kinetically homogeneous, and its kinetic properties are intermediate between those of b and a forms. The b, hybrid, and a forms of phosphorylase can be isolated from rapidly frozen muscle of crucian but in different proportions, depending on whether fish were anesthetized or forced to muscular activity for 20 s. Muscle of anesthetized crucian had 36, 36, and 28% of phosphorylase b, hybrid, and a forms, respectively, whereas the corresponding values for exercised fish were 12, 37, and 51%. Results suggest that three interconvertible forms of phosphorylase exist simultaneously in crucian muscle and that hybrid phosphorylase is active in contracting muscle in vivo.

Conference Article| August 01 1988 Glycogen phosphorylase in fish brain (Carassius carassius) during hypoxia HARALD SCHMIDT; HARALD SCHMIDT 1Institut für Zoologie, Johannes-Gutenberg-Universität, Saarstrasse 21, D 6500 Mainz, F.R.G. Search for other works by this author on: This Site PubMed Google Scholar GERHARD WEGENER GERHARD WEGENER 1Institut für Zoologie, Johannes-Gutenberg-Universität, Saarstrasse 21, D 6500 Mainz, F.R.G. Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1988) 16 (4): 621–622. https://doi.org/10.1042/bst0160621 Article history Received: November 25 1987 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn Email Cite Icon Cite Get Permissions Citation HARALD SCHMIDT, GERHARD WEGENER; Glycogen phosphorylase in fish brain (Carassius carassius) during hypoxia. Biochem Soc Trans 1 August 1988; 16 (4): 621–622. doi: https://doi.org/10.1042/bst0160621 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search This content is only available as a PDF. © 1988 Biochemical Society1988 Article PDF first page preview Close Modal You do not currently have access to this content.

The enzyme is made up from subunits ofMr-81600, and the smallest catalytically active form is likely to be a tetramer.