Lipoprotein lipase activity in the flight muscle of Locusta migratoria and its specificity for haemolymph lipoproteins

Lipoprotein lipase activity in flight muscle homogenates of Locusta migratoria was measured, using natural radiolabelled lipoproteins as substrates. The flight specific lipoprotein A⁺ (or low density lipophorin) stimulated lipoprotein lipase activity several-fold compared to the resting lipoprotein A_y (or high density lipophorin). However, with the high mol. wt lipoprotein fraction O_AKH as a substrate, lipase activity was even doubled compared to lipoprotein A⁺. Lipase activity was not increased in flight muscle homogenates of insects which had flown. Neither adipokinetic hormone, nor octopamine had any direct effect on lipoprotein lipase activity. Aspects of hormonal regulation and apoprotein activation of the locust flight muscle lipoprotein lipase are discussed and compared with the model for vertebrate lipoprotein lipase.     

Locust haemolymph lipoproteins visualised in the electron microscope

Summary In matureLocusta, the haemolymph lipoproteins change both in quality and quantity according to the physiological state of the animal. In resting locusts the majority of lipid in the haemolymph is carried by lipoprotein A_yellow, but during flight or after adipokinetic hormone injection, A_yellow joins together with extra diacylglycerols from the fat body and non-lipid carrying C_L-proteins to form another lipoprotein, A⁺. In this study partially purified A_yellow and A⁺ lipoproteins have been visualised by transmission electron microscopy after negative staining or shadowing. Both A_yellow and A⁺ lipoproteins are discrete particulate structures but they differ markedly in size; A_yellow particles are 9–16 nm in diameter while those of A⁺ are mostly in the range 20–50 nm. Large lipoprotein particles of the A⁺ type have not been described previously in insect haemolymph but, interestingly, the locust A⁺ particles do resemble most closely the low density lipoprotein particles described in human serum by Forte and Nichols (1972).     

Lipoprotein transformations during adipokinetic hormone action in Locusta migratoria

ABSTRACT. Detailed studies of lipoprotein A⁺ formation during AKH action have been made in Locusta migratoria L. using gel filtration combined with the use of radiolabeled haemolymph protein probes. In addition, we have assessed the quantitative contribution of the C_L-proteins to A⁺ formation by direct measurement of the changes in concentration of free C_L-proteins and report some properties of the C-I and C-II proteins: they appear to be glycoproteins of 20,000 and 16,000 MW respectively, but do not bind to concanavalin A. We have confirmed earlier observations (using different techniques) which showed that liproprotein A_yellow is not involved per se in A⁺ formation during the first 15 min of AKH action. In contrast, the (two) C_L-proteins take part in A⁺ formation without any apparent delay after hormone injection. Our observations show that A⁺ formation is essentially complete within 30 min of AKH injection, although further C_L-protein binding and lipid-loading do occur subsequently. After 30 min there is no further decrease in the A_yellow titre. It is argued that much, if not all, C_L-protein is located at the surface of the A⁺ particle. From the changes in titres which occur in A_yellow and C_L-proteins during AKH action we estimate that A⁺ is formed from 1 mole of A_yellow and approximately 28 moles of C_L-proteins. Using these figures we calculate an apparent molecular weight for A⁺ within the range of 1.65–2.12×10⁶, which is in reasonable agreement with estimates derived from gel exclusion chromatography data. These studies emphasize the dynamic and fully reversible nature of lipoprotein A⁺ formation and highlight the complex nature of the lipoprotein transformations occurring during hormone-stimulated lipid transport in locusts.     

Protein-lipoprotein interactions in the haemolymph of Locusta during the action of adipokinetic hormone: The role of CL-proteins

The involvement of C_L-proteins in the formation of lipoprotein A⁺ during adipokinetic hormone action has been investigated using radiolabelling experiments. Injected [³H]-C_L-proteins associate rapidly with lipoprotein A⁺ during its formation. Both [³H]-C_L-proteins and [³H]-Ayellow are liberated from [³H]-A⁺ during its natural degradation in the haemolymph (when adipokinetic hormone action is declining). It appears that [³H]-C_L-proteins bind reversibly to A⁺, since they are easily displaced in vivo and in vitro by competing concentrations of non-labelled C_L-proteins. It is suggested that Ayellow is an integral component of the A⁺ lipoprotein complex, whereas C_L-proteins may play only a relatively minor part in its structural organisation. Possible functions of the binding of C_L-proteins to A⁺ are discussed.     

In vitro studies on hormone-stimulated lipid mobilization from fat body and interconversion of haemolymph lipoproteins of Locusta migratoria

Both adipokinetic hormone and octopamine have a stimulating effect on lipid release from locust fat body in vitro, when incubated in diluted haemolymph. The presence of adipokinetic hormone results in the formation of the flight-specific haemolymph lipoprotein A⁺ accepting the increased amount of lipids released into the incubation medium. In contrast, interconversions of lipoproteins do not occur when octopamine is added to the incubation medium, which is in line with the expectations: the lipid-mobilizing effect of octopamine is a limited and short-term effect. When fat body tissue is incubated with isolated haemolymph protein fractions, the lipid-mobilizing effect of adipokinetic hormone only occurs when the incubation medium contains both lipoprotein, A_y and protein fraction C, resulting in the formation of lipoprotein A⁺. In similar control incubations with the hormone omitted, some lipoprotein A⁺ is also formed (concomitant with a slight amount of lipid released), though significantly less than in incubations with hormone. Besides a stimulating function on lipolytic processes in the fat body, adipokinetic hormone is suggested to influence haemolymph lipoprotein rearrangement. A possible counteracting function of another factor in the haemolymph is discussed.     

Qualitative and quantitative changes in Locusta haemolymph proteins and lipoproteins during ageing and adipokinetic hormone action

Changes in haemolymph proteins and lipoproteins during adipokinetic hormone action have been studied using polyacrylamide gel electrophoresis (PAGE) and a heparin/EDTA precipitation technique. During hormone action, the formation of A⁺ takes place at the expense of Ayellow and C_L-proteins, which decrease in free concentration in the haemolymph. Ayellow is heparin precipitable, whereas A⁺ precipitates with EDTA after prior treatment with heparin. After injection of adipokinetic hormone, heparin-precipitable protein (HPP) decreases after a delay of 10–15 min, but heparin/EDTA precipitable protein (HEPP) increases immediately. These changes occur in response to extracts of corpora cardiaca and to synthetic adipokinetic hormone, and are dose-dependent. Both the lipid and the C_L-protein content of the HEPP rise as its protein content increases. A⁺ formation does not occur in fifth-instar nymphs or newly emerged adults, but this response to adipokinetic hormone develops slowly as the adults mature.     

Interrelationships between haemolymph lipid and carbohydrate during starvation in Locusta

During starvation in adult female Locusta, the haemolymph total lipid concentration increases markedly while that of the total carbohydrate decreases. The majority of the increased haemolymph lipid is diglyceride and 75% of this is associated with a high molecular weight lipoprotein (A⁺) which disappears rapidly after feeding when the total lipid concentration is restored to the normal resting value. The effect of feeding can be mimicked by injecting or feeding starved locusts with sugars but not with protein. The lowering of the haemolymph total diglyceride concentration in starved locusts by injection of carbohydrates is dose-related and, at doses in excess of 4 mg per locust, almost normal values (for fed locusts) are obtained within 6 hr. It is suggested that in the haemolymph there is an inverse relationship between the concentration of diglyceride and that of trehalose.     

Diglyceride-transporting lipoproteins inLocusta

Summary The majority of haemolymph proteins obtained from resting mature locusts are soluble in 50% saturated (NH₄)₂SO₄ solution. Chromatographically, these proteins may be resolved on Sepharose 6B into three partially included fractions. The leading high molecular weight fraction, A, is predominant and all the diglyceride in resting blood is associated with it. An additional higher molecular weight fraction, A⁺, is present in the haemolymph of locusts injected with extracts of the glandular lobes of the corpora cardiaca (elevated blood), and this fraction carries all the increased diglyceride. Fraction A⁺ is better resolved on Ultrogel ACA 22. A useful rapid technique for studying the formation of A⁺ lipoprotein during lipid mobilization has been developed by the use of lipoproteinpolyanion-metal ion complex formation. A⁺ is soluble after heparin treatment whereas the yellow leading edge of fraction A precipitates. Concomitant with the appearance of A⁺ there is a marked depletion of a lower molecular weight non lipid-carrying protein fraction, C. The ability to form fraction A⁺ after injections of glandular lobe extract cannot be demonstrated in fledglings (immature locusts within 2 or 3 days after the imaginal moult) where fraction C is virtually absent. It is suggested that during lipid mobilization, some lipoprotein from fraction A combines with protein from fraction C to form A⁺. A direct effect of adipokinetic hormone on the process of A⁺ formation cannot be excluded.     

Adipokinetic hormone-induced lipid mobilization and lipophorin interconversions in fifth larval instar locusts

The response of fifth larval instar locusts to injected adipokinetic hormone (AKH) is only poor, as is reflected in both a very moderate elevation of the haemolymph lipid concentration and the slight occurrence of the haemolymph lipophorin interconversions characteristic for adult locusts, resulting in formation of only small quantities of the low density lipophorin (A⁺). However, an additional lipophorin fraction (A′) is induced, which is intermediate in density and size between high and low density lipophorin and which is not identified in adult haemolymph. As in adults, larval A⁺ formation includes association of the resting high density lipophorin with a non-lipid containing protein (C₂), the haemolymph concentration of which is only one-fifth relative to adults. However, the larval haemolymph protein composition is not the primary cause of the incomplete adipokinetic response, as elevation of the concentration of protein C₂ by injection of isolated adult C₂, whether or not in combination with adult high density lipophorin, did not increase lipophorin conversions nor haemolymph lipid elevation.In vitro incubation of larval fat bodies in adult haemolymph showed that competency to both the AKH-induced lipid release and the haemolymph lipophorin conversions of the larval fat body are reduced compared to equal amounts of adult tissue. Reciprocal incubation of adult fat body in larval haemolymph resulted in only a very moderate adipokinetic response, demonstrating that larval haemolymph protein composition is restrictive for full development of hormone action.Both immunoblotting experiments and enzyme-linked immunosorbent assays (ELISA), using monoclonal antibodies specific for the adult lipophorin apoproteins, indicated that the larval lipophorins closely resemble the adult forms. Apparently the structure of locust lipophorins is remarkably constant throughout development despite changes in metabolic functions.     

Diacylglycerol-transporting lipoproteins and flight in Locusta

Evidence from chromatographic and heparin precipitation studies shows that the ‘heparin-soluble’ lipoprotein, A⁺, forms in the haemolymph during flight. In locusts flown continuously for 60 min, lipoprotein A⁺ occurs in the haemolymph at low concentrations but accumulates during a short rest period following flight. After injections of tissue extracts containing adipokinetic hormone (AKH), A⁺ accumulates in the haemolymph but disappears more rapidly in flying locusts than in resting locusts. This difference in the rate of disappearance of diacylglycerol from the lipoprotein A⁺ can be used to estimate its rate of utilization during sustained flight (approx. 100μg. min^?1 from 45–90 min of flight). It is suggested that lipoprotein A⁺ is the major carrier of diacylglycerol from the fat body to the flight muscles during prolonged flight. The steady state concentrations of total diacylglycerol and ‘heparin-soluble’ diacylglycerol during continuous flight are unaffected when tissue extracts containing AKH are injected before flight. This suggests that there is a close homeostatic control over the steady state concentration of haemolymph lipid during flight.     

The recycling of protein components of the flight-specific lipophorin in Locusta migratoria

The metabolism of locust lipophorin A⁺ during lipid delivery to the flight muscle and lipid loading at the fat body was studied in vitro. Protein C₂ was shown to be released upon hydrolysis of lipophorin A⁺-carried diacylglycerol by the flight muscle lipoprotein lipase. This in vitro released protein C₂ was shown to reassociate with lipophorin A_y upon hormone-induced lipid mobilization from fat body in vitro. These results demonstrate the reversibility of the association of protein C₂ with lipophorin A_y and support the shuttle function of the protein components of locust lipophorin A⁺ in lipid transport.     

5-hydroxytryptamine regulates the (Na++K+)ATPase activity in malpighian tubules of Rhodnius prolixus: Evidence for involvement of G-protein and cAMP-dependent protein kinase

5-Hydroxytryptamine (5-HT, serotonin) acts as a diuretic hormone in Rhodnius prolixus, where it increases to 0.1 μM in the haemolymph during feeding and stimulates the fluid secretion in isolated Malpighian tubules. The ouabain-sensitive (Na⁺+K⁺)ATPase activity present in homogenates of Malpighian tubules from unfed Rhodnius prolixus is inhibited 60% by 0.01 μM 5-HT. This inhibition is reversed by ketanserin, a 5-HT₂ receptor antagonist in mammals, and also by GDPβS, a competitive inhibitor of G-protein GTPase activity. GTPγS, a nonhydrolysable analog of GTP, and cholera toxin, a G_s-protein activator, also inhibit the ouabain-sensitive (Na⁺+K⁺)ATPase activity, while pertussis toxin, a G_i-protein inhibitor, has no effect. The (Na⁺+K⁺)ATPase activity is inhibited 55% by 0.4–100 μM dibutyryl-cAMP in the presence of IBMX, a phosphodiesterase inhibitor, which also potentiates the effect of a low concentration of 5-HT. The cAMP-dependent protein kinase inhibitor peptide abolishes the 5-HT effect. These data suggest that the (Na⁺+K⁺)ATPase activity in Malpighian tubules is inhibited by 5-HT through activation of G_s-protein and a cAMP-dependent protein kinase. Inhibition of the Na⁺+K⁺ pump would contribute to the diuretic effect of 5-HT. Arch. Insect Biochem. Physiol. 36:203–214, 1997. © 1997 Wiley- Liss, Inc.     

Water-soluble glycoproteins from Cannabis sativa (South Africa)

The water-soluble glycoproteins obtained from Cannabis leaves of plants grown from South African seeds have been further studied. Treatment of the glycoprotein fractions with NaOH in the presence of NaBH₄, resulted in a significant decrease in the serine content and a corresponding increase in alanine. The carbohydrate side chains released contained the sugar alcohol, galactitol. By treatment of the glycoprotein fractions with NaOH in the presence of Na₂SO₃, and subsequent acid hydrolysis, cysteic acid was formed. These data indicate that carbohydrate and protein are connected via serine-O-galactoside linkages. Further investigation of the structure of the carbohydrate part of the glycoproteins was carried out by methylation analysis, Smith-degradation and enzyme incubation. The present glycoprotein material of plants grown from South African seeds is similar to the material previously investigated, but in contrast to the latter, it is devoid of hexosamine.     

In vitro and in vivo effects of copper on haemocyanin-o2 binding in the shore crab,Carcinus maenas

1. When added in vitro to crab haemolymph at concentrations of 50 or 100 mg.1⁻¹, copper decreased both haemocyanin-O₂ affinity and the cooperativity of O₂ binding.2. In crabs contaminated by a lethal dose of waterborne copper (2mg.l⁻¹), haemolymph total concentration of the metal never reached levels that could affect O₂ binding properties directly.3. Exogenous copper added in vitro or entering the animal in contaminated water was found for the most part non filterable and thus probably bound to haemolymph proteins.     

Studies of the morphology of chromosomes of some selected species

Karyotypes of twelve species from twenty-four localities in southern Moravia and one locality in Slovakia were investigated. Their counts or karyotypic formulae are as follows:Chenopodium foliosum (Moench) Ascherson: K (2n)=18=16 A^m+2 B^sm;Astragalus austriacus Jacq.: K (2n)=16=8 A^m+8 B^sm;Astragalus exscapus L.: K (2n)=16=10 A^m+4 B^sm+2 C^st;Astragalus cicer L.: K (2n)=64;Astragalus onobrychis L.: K (2n=64 and K (2n)=64+1;Vicia dumetorum L.: K (2n=14=10 A^m+4 B^sm;Vicia sylvatica L.: K (2n)=14=2 A^m+10 B^sm+2 C^st;Vicia pisiformis L.: K (2n)=12=8 A^m+4 B^sm;Vicia cassubica L.: K (2n)=12=4 A^m+6 B^sm+2 C^st;Vicia cracca L. (from five localities in southern Moravia): K (2n)=28=4 A^m+12 B^sm+12 C^st and K (2n)=28+1=5 A^m+12 B^sm+12 C^st;Vicia cracca L. (from one locality in Slovakia): K (2n)=14=2 A^m+6 B^sm+6 C^st;Vicia tenuifolia Roth: K (2n)=24=4 A^m+16 B^sm+4 C^st;Serratula lycopifolia (Vill.) Kern.: K (2n)=60.     

Haemolymph protein patterns during the spinning stage and metamorphosis of Rhynchosciara americana

An acrylamide gel electrophoretical analysis of the haemolymph proteins of R. americana was carried out at different stages of development. In mature larvae there are about 14 haemolymph protein fractions from which one stains heavily and two others faintly for lipoprotein, while three fractions stain for glycoprotein. The haemolymph protein fraction with Rm 0·25 decreases remarkably in mass during spinning, while the others decrease to a lesser extent. The protein fractions could be used in cocoon spinning since previous work suggests that haemolymph proteins are a major pool of cocoon protein precursors. The finding of a protein fraction in the salivary glands with an electrophoretical mobility similar to that of the haemolymph fraction with Rm 0·25 reinforces our hypothesis.     

Feeding impairs chill coma recovery in the migratory locust (Locusta migratoria)

Low temperature causes loss of neuromuscular function in a wide range of insects, such that the animals enter a state known as chill coma. The ability to recover from chill coma (chill coma recovery time) is often a popular phenotype to characterise chill tolerance in insects. Chill coma in insects has been shown to be associated with a decrease in haemolymph volume and a marked increase in [K⁺], causing dissipation of K⁺ equilibrium potential and resting membrane potential. High potassium diet (wheat) has also previously been shown to increase haemolymph [K⁺] in Locusta migratoria leading to sluggish behaviour. The present study combined these two independent stressors of ion and water homeostasis, in order to investigate the role of K⁺- and water-balance during recovery from chill coma, in the chill sensitive insect L. migratoria. We confirmed that cold shock elicits a fast increase in haemolymph [K⁺] which is likely caused by a water shift from the haemolymph to the muscles and other tissues. Recovery of haemolymph [K⁺] is however not only reliant on recovery of haemolymph volume, as the recovery of water and K⁺ is decoupled. Chill coma recovery time, after 2 h at −4 °C, differed significantly between fasted animals and those fed on high K⁺ diet. This difference was not associated with an increased disturbance of haemolymph [K⁺] in the fed animals, instead it was associated with a slowed recovery of muscle [K⁺], muslce water, haemolymph [Na⁺] and K⁺equilibrium potential in the fed animals.     

Dynamics of energy substrates in the haemolymph of Locusta migratoria during flight

In the two-fuel system for flight of the migratory locust, the haemolymph carbohydrate concentration falls during flight periods of up to 1 hr, the decrease being greater in case the pre-flight carbohydrate level is higher. The increase in the lipid concentration from the onset of flight is virtually independent of the initial lipid concentration. Flight intensity affects these changes in substrate concentrations: the carbohydrate level decreases more rapidly if flight speed is higher, whereas the increase in lipid concentration is delayed at higher flight speeds. Respiratory carbon dioxide production is elevated rapidly during flight and reaches over eight times the resting level. From the rate of ¹⁴CO₂ production after labelling of the haemolymph diglyceride pool it is concluded that diglycerides contribute to providing the energy for flight from the earliest stage of flying activity; diglyceride oxidation increases until maximum utilization is attained after some 45 min of flight. The decline in haemolymph carbohydrate concentration due to flying activity results in a decrease of haemolymph osmolarity. Free amino acids, particularly taurine, increase markedly in the haemolymph during flight; yet their concentration only partially counterbalances the fall in haemolymph osmolarity.     

Identification of Microbial Communities in Open and Closed Circuit Bioelectrochemical MBRs by High-Throughput 454 Pyrosequencing

Two bioelectrochemical membrane bioreactors (MBRs) developed by integrating microbial fuel cell and MBR technology were operated under closed-circuit and open-circuit modes, and high-throughput 454 pyrosequencing was used to investigate the effects of the power generation on the microbial community of bio-anode and bio-cathode. Microbes on the anode under open-circuit operation (A_O) were enriched and highly diverse when compared to those on the anode under closed-circuit operation (A_C). However, among the cathodes the closed-circuit mode (C_C) had richer and more diverse microbial community compared to the cathode under open-circuit mode (C_O). On the anodes A_O and A_C, Proteobacteria and Bacteroidetes were the dominant phyla, while Firmicutes was enriched only on A_C. Deltaproteobacteria affiliated to Proteobacteria were also more abundant on A_C than A_O. Furthermore, the relative abundance of Desulfuromonas, which are well-known electrogenic bacteria, were much higher on A_C (10.2%) when compared to A_O (0.11%), indicating that closed-circuit operation was more conducive for the growth of electrogenic bacteria on the anodes. On the cathodes, Protebacteria was robust on C_C while Bacteroidetes was more abundant on C_O. Rhodobacter and Hydrogenophaga were also enriched on C_C than C_O, suggesting that these genera play a role in electron transfer from the cathode surface to the terminal electron acceptors in the bioelectrochemical MBR under closed-circuit operation.     

Glycolipid- and glycoprotein-based blood group A antigen expression in human thrombocytes. A1/A2 difference

Total non-acid glycolipid fractions and total sodium dodecylsulphate (SDS) solubilized protein fractions were isolated from human thrombocytes obtained from single human donors having different blood group A₁/A₂ phenotypes. The blood group A glycolipid antigens were characterized by immunostaining of thin layer plates with different monoclonal anti-A antibodies. The glycoproteins carrying blood group A epitopes were identified by SDS-PAGE and Western blot analysis using a monoclonal anti-A antibody. Blood group A glycolipid antigens were found in both A₁ and A₂ thrombocytes but the A₂ individuals expressed at least ten times less A glycolipids compared to the A₁ individuals. Expression of A type 3/4 chain and small amounts of A type 1 chain glycolipids were seen in thrombocytes of both A₁ and A₂ individuals, while the type 2 chain A glycolipids appeared to be missing from the A₂ thrombocytes. Blood group A reactive glycoproteins were only found in thrombocytes of A₁ individuals and could not be detected in A₂ individuals or a blood group O individual. The major blood group A glycoprotein were found as a double band migrating in the 130 kDa region.Abbreviations SDS sodium dodecyl sulfate - PAGE polyacrylamide gel electrophoresis - HPTLC high performance thin layer chromatography - CBB Coomassie brilliant blue - GVH graft versus host Part of this work was presented at the Xth International Symposium on Glycoconjugates, Jerusalem, Israel. September, 1989.In the short hand designation for glycolipids, the letter indicate blood group determinant, the first numeral, the number of sugar residues, and the second numeral, the type of carbohydrate chain. Thus, A-6-1 means a hexaglycosylceramide with a blood group A determinant based on the type 1 carbohydrate chain.