Amino acid metabolism during flight in tsetse flies.

Experiments carried out using teneral Glossina pallidipes indicate that flight can continue for at least 4 to 7 min after the thoracic proline reserves have fallen to low levels, suggesting that some other energy source is available. Earlier work suggests that alanine formed during flight is transported from the thorax to the abdomen where proline is resynthesized. Injection experiments using ¹⁴C alanine confirm that the transport mechanism does occur, that it is enhanced by flight, and that alanine is more rapidly incorporated into glutamate and proline in the abdomen than in the thorax. An analysis of published work shows that there is evidence for the involvement of residual blood meal amino acids even in the early stages of flight and supports the suggestion that they are of importance in prolonging flight. A decline in amino nitrogen during the early stages of flight is consistent with the action of glutamate dehydrogenase at this time. The poor flight durations in teneral flies may be due both to the low proline levels and to the absence of the residual blood meal. Very high energy consumptions are noted and appear to be related to the abnormally large musculature necessary for the fly's haematophagous and viviparous habits.     

Isolation and properties of flight muscle mitochondria of the bumblebee Bombus terrestris (L.)

This report describes the isolation procedure and properties of tightly coupled flight muscle mitochondria of the bumblebee Bombus terrestris (L.). The highest respiratory control index was observed upon oxidation of pyruvate, whereas the highest respiration rates were registered upon oxidation of a combination of the following substrates: pyruvate + malate, pyruvate + proline, or pyruvate + glutamate. The respiration rates upon oxidation of malate, glutamate, glutamate + malate, or succinate were very low. At variance with flight muscle mitochondria of a number of other insects reported earlier, B. terrestris mitochondria did not show high rates of respiration supported by oxidation of proline. The maximal respiration rates were observed upon oxidation of α-glycerophosphate. Bumblebee mitochondria are capable of maintaining high membrane potential in the absence of added respiratory substrates, which was completely dissipated by the addition of rotenone, suggesting high amount of intramitochondrial NAD-linked oxidative substrates. Pyruvate and α-glycerophosphate appear to be the optimal oxidative substrates for maintaining the high rates of oxidative metabolism of the bumblebee mitochondria.     

Flight metabolism in the African fruit beetle,Pachnoda sinuata

Metabolite concentrations in flight muscles and in abdomen of beetles (Pachnoda sinuata) were measured after various periods of tethered flight and subsequent rest. Three distinct phases of energy metabolism are found in active flight muscles: (1) during the first minutes of flight proline is used as main substrate and concomitantly alanine accumulated as an end product; (2) the second phase is characterized by a large-scale degradation of glycogen; (3) after about 8 min of flight the metabolite levels stabilize, while flight performance appears unchanged. After the termination of flight the preflight proline concentration (70 mol·g^-1 fw) is re-established in less than 60 min, whereas restoration of resting levels of other metabolites requires longer. The pattern of maximal enzyme activities and the respiratory rates of mitochondria with different substrates confirm the significance of proline and carbohydrates as the main fuels of working flight muscles.Abbreviations CS citrate synthetase - Cytox cytochrome c oxidase - EDTA ethylenediaminetetra-acetate - fw fresh weight - GluDH glutamate dehydrogenase - GPT alanine aminotransferase - HOAD hydroxyacyl-coenzyme A dehydrogenase - HPLC high pressure liquid chromatography - ME malic enzyme - PCA perchloric acid - RQ repiratory quotient - TRA triethanolamine     

The nature and control of the tricarboxylate cycle in beetle flight muscle.

The only exogenous substrates oxidized by mitochondria isolated from the flight muscle of the Japanese beetle (Popillia japonica) are proline, pyruvate and glycerol 3-phosphate. The highest rate of oxygen consumption is obtained with proline. The oxidation of proline leads to the production of more NH3 than alanine, indicating a functioning glutamate dehydrogenase (EC 1.4.1.2). Studies of mitochondrial extracts confirm the presence of a very active glutamate dehydrogenase, and this enzyme is found to be activated by ADP and inhibited by ATP. These extracts also show high alanine aminotransferase activity (EC 2.6.1.2) and a uniquely active "malic'' enzyme (EC 1.1.1.39). The "malic'' enzyme is activated by succinate and inhibited by ATP and by pyruvate. It is suggested that the input of tricarboxylate-cycle intermediate from proline oxidation is balanced by the formation of pyruvate from malate, and the complete oxidation of the majority of the pyruvate. Studies of the steady-state concentrations of mitochondrial CoASH and CoA thioesters during proline oxidation show a high succinyl (3-carboxypropionyl)-CoA content which falls on activating respiration with ADP. There is a concomitant rise in CoASH. However, the reverse transition, from state-3 to state-4 respiration, causes only very slight changes in acylation. The reasons for this are discussed. Studies of the mitochondrial content of glutamate, 2-oxoglutarate, malate, pyruvate, citrate and isocitrate during the same phases of proline oxidation give results consistent with control at the level of glutamate dehydrogenase and isocitrate dehydrogenase during proline oxidation, with the possibility of further control at "malic'' enzyme. During the oxidation of pyruvate all of the tricarboxylate-cycle intermediates and NAD(P)H follow the pattern of changes described in the blowfly (Johnson & Hansford, 1975; Hansford, 1974) and isocitrate dehydrogenase is identified as the primary site of control.?2OAuthor     

Fuels for energy metabolism in the Colorado potato beetle, Leptinotarsa decemlineata Say

During flight, the proline concentration in the flight muscles of Leptinotarsa decemlineata decreases sharply while that of alanine shows a proportional increase. In view of the known dynamics of proline turn over the observed decrease would indicate that proline plays the major role as a mobilizable energy source for flight. To a minor extent carbohydrates such as glycogen and glucose support energy metabolism during flight in the Colorado potato beetle.During a recovery period after flight the proline concentration in the fat body increases sharply within minutes, a feature which would indicate that fat body could synthesize proline maybe at the expense of alanine. Comparison of starvation and flight metabolism reveals that the metabolic changes in the two situations are different.     

Proline can be utilized as an energy substrate during flight of Aedes aegypti females

In order to determine whether proline can be utilized as fuel during flight of Aedes aegypti, proline, alanine, and glutamine concentrations were monitored at 0, 30 and 60 min after flight using sugar-fed males and females, and blood meal-fed females. In sugar-fed and blood meal-fed females, flight lead to a significant decrease in proline and a significant increase in glutamine concentration in both hemolymph and thorax. Only during flight after a blood meal was a significant increase in the alanine concentration observed in hemolymph. After flight, the proline alanine and glutamine levels in the hemolymph and thorax from males did not change significantly. In addition, activities of enzymes related to amino acid metabolism were assayed in homogenates of cephalothorax and thorax from both sexes, and in fat body and midgut from females. In both sexes, the activities of all the enzymes studied were significantly higher in thorax than in cephalothorax. The levels of the enzymes involved in proline oxidation were higher in thorax than in fat body and midgut. These results suggest that proline can be used as an energy substrate for flight muscle of Ae. aegypti females. However, the elevation in glutamine levels observed in hemolymph and thorax after flight has not been reported in other insects that fuel flight using proline and may suggest an additional mechanism for shuttling ammonia between flight muscle and fat body is present in mosquitoes.     

Beetles' choice--proline for energy output: control by AKHs

Many beetle species use proline and carbohydrates in a varying ratio to power flight. The degree of contribution of either fuel varies widely between species. In contrast, dung beetle species investigated, thus far, do not have any carbohydrate reserves and rely completely on proline to power energy-costly activities such as flight and, probably, walking and ball-rolling. While the fruit beetle, Pachnoda sinuata, uses proline and carbohydrates equally during flight, proline is solely oxidised during endothermic pre-flight warm-up, as well as during flight after prolonged starvation. Thus, proline seems to be the essential fuel for activity in beetles, even in flightless ones and in those that use proline in combination with carbohydrates; the latter can be completely substituted by proline in certain circumstances. It is apparent from the rapid decline of energy substrates in flight muscles and haemolymph after the onset of flight that mobilisation of stored fuels of the fat body is necessary for prolonged flight periods. This task is performed by AKH-type neuropeptides. In beetles, like in other insects, these peptides mobilise glycogen via activation of glycogen phosphorylase. They also stimulate proline synthesis from alanine and acetyl-CoA in the fat body. Acetyl-CoA is derived from the beta-oxidation of fatty acids and we propose that the neuropeptides activate triacylglycerol lipase.     

Importance of proline and other amino acids during honeybee flight

Summary. The levels of proline and other amino acids in the haemolymph and other body parts of honeybee foragers were investigated by HPLC analysis. The concentrations of proline in the blood of glucose-fed or -injected bees finishing their exhaustive tethered flights on a roundabout were significantly reduced compared to bees that were fed and rested for one hour. This indicates some utilization of proline during flight metabolism. The levels of essential amino acids and of the sum of all amino acids except proline remained roughly constant, indicating that the decrease of proline did not result from a changed haemolymph volume. ¹⁴C-labelled proline was injected into bees either shortly before starting their flight or before a resting period of equal duration in an incubator at the same temperature. Bees that rested had incorporated more proline into thorax body protein, and less of the labelled substance was unrecovered ("missing") and considered to be respired or less probably defecated. If the entire amount of missing ¹⁴C-proline is regarded as exhaled, the oxidative breakdown of proline reached higher levels after flight than in rested bees. This is another hint that proline is utilized during flight. Usually the exhaled amount did not exceed 10 μg proline in half an hour of flight. Although our data indicate involvement of proline in flight metabolism, the amount metabolized is low compared to the utilization of carbohydrates. Received December 5, 1998, Accepted February 1, 1999     

Metabolism of arginine in lactating rat mammary gland.

Significant activities of the four enzymes needed to convert arginine into proline and glutamate (arginase, ornithine aminotransferase, pyrroline-5-carboxylate reductase and pyrroline-5-carboxylate dehydrogenase) develop co-ordinately in lactating rat mammary glands in proportion to the increased production of milk. No enzymes were detected to carry out the reactions of proline oxidation or reduction of glutamate to pyrroline-5-carboxylate. Minces of the gland converted ornithine into proline and into glutamate plus glutamine. These conversions increased during the cycle of lactation in proportion to the increased milk production and to the content of the necessary enzymes. The minced gland did not convert labelled ornithine into citrulline, confirming the absence from the gland of a functioning urea cycle, and did not convert labelled proline or glutamate into ornithine. A metabolic flow of labelled arginine to proline and glutamate in mammary gland was confirmed in intact animals with experiments during which the specific radioactivity of proline in plasma remained below that of the proline being formed from labelled arginine within the gland. It was concluded that arginase in this tissue had a metabolic role in the biosynthesis of extra proline and glutamate needed for synthesis of milk proteins.     

Insights into the roles of exogenous glutamate and proline in improving streptolydigin production of Streptomyces lydicus with metabolomic analysis

The addition of precursors was one strategy to improve antibiotic production. The exogenous proline and glutamate, as precursors of streptolydigin, could significantly improve the streptolydigin production, but their underlying molecular mechanisms remain unknown. Herein, metabolomic analysis was carried out to explore the metabolic responses of Streptomyces lydicus to the additions of proline and glutamine. The significant differences in the quantified 53 metabolites after adding the exogenous proline and glutamate were enunciated by gas chromatography coupled to time-of-flight mass spectrometry. Among them, the levels of some fatty acids (e.g., dodecanoic acid, octadecanoic acid, hexadecanoic acid) were significantly decreased after adding glutamate and proline, indicating that the inhibition of fatty acid synthesis might be benefit for the accumulation of streptolydigin. Particularly, the dramatic changes of the identified metabolites, which are involved in glycolysis, the tricarboxylic acid cycle, and the amino acid and fatty acid metabolism, revealed that the additions of glutamate and proline possibly caused the metabolic cross-talk in S. lydicus. Additionally, the level of intracellular glutamate dramatically enhanced at 12 h after adding proline, showing that exogenous proline may be firstly convert into glutamate and consequently result in crease of the streptolydigin production. The high levels of streptolydigin at 12 and 24 h after adding glutamate unveiled that part glutamate were rapidly used to synthesize the streptolydigin. Furthermore, there is the significant difference in metabolomic characteristics of S. lydicus after adding glutamate and proline, uncovering that multiple regulatory pathways are involved in responses to the additions of exogenous glutamate and proline. Taken together, exogenous glutamate and proline not only directly provided the precursors of streptolydigin biosynthesis, but also might alter the metabolic homeostasis of S. lydicus E9 during improving the production of streptolydigin.     

Energy coupling in the active transport of proline and glutamate by the photosynthetic halophile Ectothiorhodospira halophila.

When illuminated, washed cell suspensions of Ectothiorhodospira halophila carry out a concentrative uptake of glutamate or proline. Dark-exposed cells accumulate glutamate but not proline. Proline transport was strongly inhibited by carbonylcyanide-m-chlorophenylhydrazone (CCCP), a proton permeant that uncouples photophosphorylation, and by 2-heptyl-4-hydroxyquinoline-n-oxide (HQNO), an inhibitor of photosynthetic electron transport. A stimulation of proline uptake was effected by N,N'-dicyclohexylcarbodiimide (DCCD), an inhibitor of membrane adenosine triphosphatase (ATPase) which catalyzes the phosphorylation. These findings suggest that the driving force for proline transport is the proton-motive force established during photosynthetic electron transport. Glutamate uptake in the light was inhibited by CCCP and HQNO, but to a lesser extent than was the proline system. DCCD caused a mild inhibition of glutamate uptake in the light, but strongly inhibited the uptake by dark-exposed cells. CCCP strongly inhibited glutamate uptake in the dark. The light-dependent transport of glutamate is apparently driven by the proton-motive force established during photosynthetic electron transport. Hydrolysis of adenosine triphosphate (ATP) by membrane ATPase apparently establishes the proton-motive force to drive the light-independent transport. These conclusions were supported by demonstrating that light- or dark-exposed cells accumulate [3H]triphenylmethylphosphonium, a lipid-soluble cation. Several lines of indirect evidence indicated that the proline system required higher levels of energy than did the glutamate system(s). This could explain why ATP hydrolysis does not drive proline transport in the dark. Membrane vesicles were prepared by the sonic treatment of E. halophila spheroplasts. The vesicles contained active systems for the uptake of proline and glutamate.     

Quantitative aspects of proline utilization during flight in tsetse flies

ABSTRACT. The proline concentration of the haemolymph in resting tsetse flies provides a reasonable indication of their total proline content. Estimates of pre-flight proline content obtained on this basis were compared with the proline content of flies that had been flown for different durations to provide an estimate of the rate of proline consumption at different stages of flight. The results indicate that the apparent ability of tsetse flies to continue flight after their proline reserves have been exhausted is an artefact of experimental procedure. It is concluded that the flight capacity of tsetse flies is effectively limited by the magnitude of their proline reserve, although this reserve is capable of being supplemented to some extent by the limited synthetic capacity of the fat body.     

Sodium-substrate cotransport in bacteria

A variety of sodium-substrate cotransport systems are known in bacteria. Sodium enters the cell down an electrochemical concentration gradient. There is obligatory coupling between the entry of the ion and the entry of substrate with a stoichiometry (in the cases studied) of 1:1. Thus, the downhill movement of sodium ion into the cell leads to the accumulation of substrate within the cell. The melibiose carrier of Escherichia coli is perhaps the most carefully studied of the sodium cotransport systems in bacteria. This carrier is of special interest because it can also use protons or lithium ions for cotransport. Other sodium cotransport carriers that have been studied recently are for proline, glutamate, serine-threonine, citrate and branched chain amino acids.     

Synteny between the pro+ marker and human glutamate oxaloacetate transaminase.

Chinese hamster ovary cells with a specific auxotrophy for proline were fused with human cells from a variety of sources and the resulting hybrids analyzed for human genetic markers. Of 63 hybrid clones examined, 27 possessed both proline and cytoplasmic glutamate oxaloacetate transaminase markers; 36 had neither; and no clones were found possessing one and not the other. These results constitute evidence that the proline and glutamate oxalocetate transaminase markers are syntenic. Evidence for absence of synteny between these and a variety of other human genes is presented. Biochemical tracer experiments established that the proline biosynthetic pathway through glutamate has been restored in the Pro+ hybrids.     

Synthesis of proline and hydroxyproline in human lung (WI-38) fibroblasts.

Human lung fibroblasts (WI-38) in late exponential phase of growth, in stationary phase after confluency was reached, and at high or low number of population doublings were used to investigate the synthesis of proline and hydroxyproline from glutamate or arginine. Glutamate was from two to five times as effective a precursor as arginine; glutamine did not seem to be involved in these metabolic pathways. Accumulation of protein-bound hydroxyproline in cell layers was observed only after confluency. Confluent cells synthesized more proline from glutamate than did cells in late exponential growth. Conversion of glutamate into intracellular free proline was conducted also to a greater extent in confluent cells at a high number of population doublings. Conversion of glutamate into proline or hydroxyproline in cell-layer protein was not affected significantly by the number of population doublings. Less total protein as well as less hydroxyproline accumulated with cells at a high number of population doublings.     

Proline promotes decrease in glutamate uptake in slices of cerebral cortex and hippocampus of rats

In the present study we first investigated the in vitro and in vivo effects of proline on glutamate uptake in the cerebral cortex and hippocampus slices of rats. The action of alpha-tocopherol and/or ascorbic acid on the effects elicited by administration of proline was also evaluated. For in vitro studies, proline (30.0 microM and 1.0 mM) was added to the incubation medium. For acute administration, 29-day-old rats received one subcutaneous injection of proline (18.2 micromol/g body weight) or saline (control) and were sacrificed 1 h later. Results showed that addition of proline in the assay (in vitro studies) reduces glutamate uptake in both cerebral structures. Administration of proline (in vivo studies) reduces glutamate uptake in the cerebral cortex, but not in the hippocampal slices of rats. In another set of experiments, 22-day-old rats were pretreated for one week with daily administration of alpha-tocopherol (40 mg/kg) or ascorbic acid (100 mg/kg) or with both vitamins. Twelve hours after the last vitamins injection, rats received a single injection of proline or saline and were killed 1 h later. Pretreatment with alpha-tocopherol and/or ascorbic acid did not prevent the effect of proline administration on glutamate uptake. alpha-Tocopherol plus ascorbic acid prevented the inhibitory effect of acute hyperprolinemia on Na(+),K(+) -ATPase activity in the cerebral cortex of 29-day-old rats. The data indicate that the effect of proline on reduction of glutamate uptake and Na(+),K(+) -ATPase activity may be, at least in part, involved in the brain dysfunction observed in hyperprolinemic patients.     

Mitochondrial transport in proline catabolism in plants: the existence of two separate translocators in mitochondria isolated from durum wheat seedlings

Abiotic stresses, such as high salinity or drought, can cause proline accumulation in plants. Such an accumulation involves proline transport into mitochondria where proline catabolism occurs. By using durum wheat seedlings as a plant model system, we investigated how proline enters isolated coupled mitochondria. The occurrence of two separate translocators for proline, namely a carrier solely for proline and a proline/glutamate antiporter, is shown in a functional study in which we found the following: (1) Mitochondria undergo passive swelling in isotonic proline solutions in a stereospecific manner. (2) Externally added l-proline (Pro) generates a mitochondrial membrane potential (ΔΨ) with a rate depending on the transport of Pro across the mitochondrial inner membrane. (3) The dependence of the rate of generation of ΔΨ on increasing Pro concentrations exhibits hyperbolic kinetics. Proline transport is inhibited in a competitive manner by the non-penetrant thiol reagent mersalyl, but it is insensitive to the penetrant thiol reagent N-ethylmaleimide (NEM). (4) No accumulation of proline occurs inside the mitochondria as a result of the addition of proline externally, whereas the content of glutamate increases both in mitochondria and in the extramitochondrial phase. (5) Glutamate efflux from mitochondria occurs at a rate which depends on the mitochondrial transport, and it is inhibited in a non-competitive manner by NEM. The dependence of the rate of glutamate efflux on increasing proline concentration shows saturation kinetics. The physiological role of carrier-mediated transport in the regulation of proline catabolism, as well as the possible occurrence of a proline/glutamate shuttle in durum wheat seedlings mitochondria, are discussed.Catello Di Martino, Roberto Pizzuto these authors contributed equally to the paper     

Role of carbohydrates in proline accumulation in wilted barley leaves

The effect of wilting on proline synthesis, proline oxidation, and protein synthesis—all of which contribute to proline accumulation—was determined in nonstarved barley (Hordeum vulgare L.) leaves. Nonstarved leaves were from plants previously in the light for 24 hours and starved leaves were from plants previously in the dark for 48 hours. Wilted leaves from nonstarved plants accumulated proline at the rate of about 1 μmole per hour per gram of fresh weight whereas wilted leaves from starved plants accumulated very little proline. Wilting caused a 40-fold stimulation of proline synthesis from glutamate in nonstarved leaves but had very little effect in starved leaves. Proline oxidation and protein synthesis, on the other hand, were inhibited by wilting in both nonstarved and starved leaves. Thus, the role of carbohydrates in proline accumulation is to supply precursors for the stimulated proline synthesis. These results further indicate that the main metabolic response causing proline to accumulate in wilted barley leaves is the stimulation of proline synthesis from glutamate. The difference between these results and those obtained with beans is discussed.

Wilting caused an increased conversion of glutamate to other products. In nonstarved leaves, conversion to organic acids as well as to proline was increased. In starved leaves, wilting caused an increase in the conversion of glutamate to glutamine, aspartate, asparagine, and organic acids.

     

The fate of proline in the African fruit beetle Pachnoda sinuata

Metabolic pathways of proline consumption in working flight muscles and its resynthesis were investigated in the African fruit beetle, Pachnoda sinuata.Mitochondria isolated from flight muscles oxidise proline, pyruvate and α-glycerophosphate, but not palmitoyl-carnitine. At low proline concentrations, the respiration rate during co-oxidation of proline and pyruvate is additive, while at high proline concentrations it is equal to the respiration rates of proline oxidation.Flight muscles have high activities of alanine aminotransferase and NAD⁺-dependent malic enzyme which are involved in proline metabolism. Glycogen phosphorylase and glyceraldehyde-3-phosphate dehydrogenase (carbohydrate breakdown) also display high activities, whilst 3-hydroxyacyl-CoA dehydrogenase (fatty acid oxidation) showed low activity.During the oxidation of proline, mitochondria isolated from flight muscles produce equimolar amounts of alanine. The rates of oxygen consumption by the mitochondria during this process lead to the conclusion that proline is partially oxidised. This is confirmed by the incorporation of radiolabel from pre-injected [U-¹⁴C] proline into alanine during a flight experiment with P. sinuata.Proline is resynthesised, in vitro, from alanine and acetyl-CoA in the fat body. High activities of enzymes catalysing such pathways (alanine aminotransferase, 3-hydroxyacyl-CoA dehydrogenase and NADP⁺-dependent malic enzyme) were found. The in vitro production of proline from alanine is equimolar suggesting that resynthesis of one proline molecule is accomplished from one alanine molecule and one acetyl-CoA molecule. One source of the acetyl-CoA for the in vitro synthesis of proline is the lipid stores of the fat body.Proline synthesis by fat body tissue is controlled by feedback. Alanine aminotransferase is competitively inhibited by high proline concentrations.