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.     

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).     

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.     

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.     

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.     

Modeling Hermissenda: I. Differential contributions of I A and I C to type-B cell plasticity

We developed a multicompartmental Hodgkin-Huxley model of the Hermissenda type-B photoreceptor and used it to address the relative contributions of reductions of two K⁺ currents, I _a and I _C, to changes in cellular excitability and synaptic strength that occur in these cells after associative learning. We found that reductions of g_C, the peak conductance of I _C, substantially increased the firing frequency of the type-B cell during the plateau phase of a simulated light response, whereas reductions of g_A had only a modest contribution to the plateau frequency. This can be understood at least in part by the contributions of these currents to the light-induced (nonspiking) generator potential, the plateau of which was enhanced by g_C reductions, but not by g_A reductions. In contrast, however, reductions of g_A broadened the type-B cell action potential, increased Ca²⁺ influx, and increased the size of the postsynaptic potential produced in a type-A cell, whereas similar reductions of g_C had only negligible contributions to these measures. These results suggest that reductions of I _A and I _C play important but different roles in type-B cell plasticity.     

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.     

Genetic and physiological evidence concerning the development of chemically sensitive voltage-dependent ionophores in L6 cells

The electrophysiological properties of a tissue culture muscle line, L₆, and a K⁺ resistant mutant (MK₁) derived from L₆ were determined to elucidate certain aspects of membrane differentiation and function. MK₁ was selected as a clone of myoblasts resistant to the toxic effects of 55 mM K⁺. The resting potentials of L₆ and MK₁ myoblasts and myotubes were K⁺ dependent and equal. The amplitudes of the action potentials were equal in normal medium, but 27.7 mM K⁺ interfered with or eliminated the ability of L₆ myotubes to produce action potentials. MK₁ myotubes produced nearly normal action potentials under these conditions. Thus, the K⁺ resistant myoblasts differentiate into myotubes which have an action potential generating mechanism much less sensitive to K⁺ than the normal mechanism. Also, both d-tubocurarine and α-bungarotoxin enhance the amplitude of the action potentials produced by L₆ myotubes in the presence of 27.7 mM K⁺; these compounds do not enhance the amplitude of the action potentials produced by MK₁ myotubes under the same conditions. It is proposed that as a consequence of differentiation a type of ionophore present in myoblasts becomes a voltage-dependent ionophore in myotubes. Furthermore, these voltage-dependent ionophores can be chemically sensitive.     

The Relation between Membrane Potential and the Transport Activity of Systems a and L in Plasma Membrane Vesicles of the Ehrlich Cell

Plasma membrane vesicles isolated from Ehrlich ascites tumor cells have been used to investigate the role of the transmembrane potential in the energetics of Systems A and L. As expected, Na⁺-dependent System A was responsive to changes in membrane potential. System L activity, as measured by transport of 2-aminonorbornane-2-carboxylic acid (BCH), was shown to be Na⁺-independent and was not altered by changes in the membrane potential. The combination of valinomycin and nigericin decreased accumulation of MeAIB but not that of BCH. The presence of nigericin alone caused a significant decrease in uptake by System A and a decrease in uptake by System L to a lesser degree. The inhibitory action of nigericin might reflect its ability to dissipate the Na⁺ gradient rather than an effect on K⁺ or H⁺ flows. The results indicate that modes of energization not produced through the transmembrane potential must account for any uphill operation of System L.     

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.     

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.     

A model of photosystem II for the analysis of fast fluorescence rise in plant leaves

The polyphasic patterns of fluorescence induction rise in pea leaves in vivo and after the treatment with ionophores have been studied using a Plant Efficiency Analyzer. To analyze in detail photosystem II (PS II) electron transfer processes, an extended PS II model was applied, which included the sums of exponential functions to specify explicitly the light-driven formation of the transmembrane electric potential (ΔΨ(t)) as well as pH in the lumen (pH_L(t)) and stroma (pH_S(t)). PS II model parameters and numerical coefficients in ΔΨ(t), pH_L(t), and pH_S(t) were evaluated to fit fluorescence induction data for different experimental conditions: leaf in vivo or after ionophore treatment at low or high light intensity. The model imitated changes in the pattern of fluorescence induction rise due to the elimination of transmembrane potential in the presence of ionophores, when ΔΨ = 0 and pH_L(t), pH_S(t) changed to small extent relative to control values in vivo, with maximum ΔΨ(t) ∼ 90 mV and ΔΨ(t) ∼ 40 mV for the stationary state at ΔpH ≅ 1.8. As the light intensity was increased from 300 to 1200 μmol m⁻² s⁻¹, the heat dissipation rate constants increased threefold for nonradiative recombination of P680⁺Phe⁻ and by ∼30% for P680⁺Q_A⁻. The parameters ΔΨ, pH_S and pH_L were analyzed as factors of PS II redox state populations and fluorescence yield. The kinetic mechanism of fluorescence quenching is discussed, which is related with light-induced lumen acidification, when ⁺Q_A⁻ and P680⁺ recombination probability increases to regulate the Q_A reduction.     

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.     

A Single mri Slice Does Not Accurately Predict Visceral and Subcutaneous Adipose Tissue Changes During Weight Loss

Earlier cross‐sectional studies found that a single magnetic resonance imaging (MRI) slice predicts total visceral and subcutaneous adipose tissue (VAT and SAT) volumes well. We sought to investigate the accuracy of trunk single slice imaging in estimating changes of total VAT and SAT volume in 123 overweight and obese subjects who were enrolled in a 24‐week CB‐1R inverse agonist clinical trial (weight change, ?7.7 ± 5.3 kg; SAT change, ?5.4 ± 4.9 l, VAT change, ?0.8 ± 1.0 l). VAT and SAT volumes at baseline and 24 weeks were derived from whole‐body MRI images. The VAT area 5–10 cm above L₄—L₅ (A_+5–10) (R² = 0.59–0.70, P < 0.001) best predicted changes in VAT volume but the strength of these correlations was significantly lower than those at baseline (R² = 0.85–0.90, P < 0.001). Furthermore, the L₄—L₅ slice poorly predicted VAT volume changes (R² = 0.24–0.29, P < 0.001). Studies will require 44–69% more subjects if (A_+5–10) is used and 243–320% more subjects if the L₄—L₅ slice is used for equivalent power of multislice total volume measurements of VAT changes. Similarly, single slice imaging predicts SAT loss less well than cross‐sectional SAT (R² = 0.31–0.49 vs. R² = 0.52–0.68, P < 0.05). Results were the same when examined in men and women separately. A single MRI slice 5–10 cm above L₄—L₅ is more powerful than the traditionally used L₄—L₅ slice in detecting VAT changes, but in general single slice imaging poorly predicts VAT and SAT changes during weight loss. For certain study designs, multislice imaging may be more cost‐effective than single slice imaging in detecting changes for VAT and SAT.     

Cloning and sequencing of cDNAs encoding two SSS

A defective S-allele, S ₀, and a functional S-allele, S _x, have previously been found to be retained in an F₁ hybrid of a self-compatible commercial cultivar of Petunia hybrida. Pistil proteins associated with these two alleles have also been identified. Their amino-terminal sequences have been found to share a high degree of similarity with those of S-proteins characterized from self-incompatible solanaceous species. Here we report the isolation and sequencing of cDNAs encoding S ₀- and S _x-proteins. Their deduced amino acid sequences contain all the consensus primary structural features of S-proteins from self-incompatible solanaceous species. Both proteins also have ribonuclease activity. The implications of these findings are discussed in relation to the presumed function of the S-protein in the self-incompatibility interaction.     

Stimulation of trophoblastic giant cell differentiation in the homozygousYellow mouse embryo

Escape from pre-implantation lethality by homozygousyellow embryos depends on differentiation of trophoblastic cells. The evidence presented herein suggests that progesterone stimulated differentiation of trophoblastic giant cells in some homozygousyellow blastocysts and thereby increased the percentage ofA ^y/A^y “escaper” embryos. This work was supported in part by funds from research grant HDOOO36, National Institute of Child Health and Development, National Institutes of Health, U.S.A.     

Adipokinetic hormone exerts its anti-oxidative effects using a conserved signal-transduction mechanism involving both PKC and cAMP by mobilizing extra- and intracellular Ca stores

The involvement of members of the adipokinetic hormone (AKH) family in regulation of response to oxidative stress (OS) has been reported recently. However, despite these neuropeptides being the best studied family of insect hormones, their precise signaling pathways in their OS responsive role remain to be elucidated. In this study, we have used an in vitro assay to determine the importance of extra and intra-cellular Ca^2 + stores as well as the involvement of protein kinase C (PKC) and cyclic adenosine 3′,5′-monophosphate (cAMP) pathways by which AKH exerts its anti-oxidative effects. Lipid peroxidation product (4-HNE) was significantly enhanced and membrane fluidity reduced in microsomal fractions of isolated brains (CNS) of Pyrrhocoris apterus when treated with hydrogen peroxide (H₂O₂), whereas these biomarkers of OS were reduced to control levels when H₂O₂ was co-treated with Pyrap-AKH. The effects of mitigation of OS in isolated CNS by AKH were negated when these treatments were conducted in the presence of Ca^2 + channel inhibitors (CdCl₂ and thapsigargin). Presence of either bisindolylmaliemide or chelyrythrine chloride (inhibitors of PKC) in the incubating medium also compromised the anti-oxidative function of AKH. However, supplementing the medium with either phorbol myristate acetate (PMA, an activator of PKC) or forskolin (an activator of cAMP) restored the protective effects of exogenous AKH treatment by reducing 4-HNE levels and increasing membrane fluidity to control levels. Taken together, our results strongly implicate the importance of both PKC and cAMP pathways in AKHs' anti-oxidative action by mobilizing both extra and intra-cellular stores of Ca^2 +.     

Molecular mechanisms of interaction of polycationic peptides with serpentine type receptors and heterotrimeric G-proteins in rat tissues

The key step in the hormonal signal transduction into cell is interaction of receptors with heterotrimeric G-proteins. We and other authors have shown that G-proteins may be activated as a result of their direct interaction with polycationic peptides. The goal of this work was to study molecular mechanisms of effect of hydrophobic peptide I, C-εAhx-WKK(C₁₀)-KKK(C₁₀)-KKKK(C₁₀)-YKK(C₁₀)-KK, and branched peptide II, [(GRGDSGRKKRRQRRRPPQ)₂-K-εAhx-C]₂ including the 48–60 fragment of the HIV-1 TAT-protein, on receptor and G-protein. These two peptides (10^?6?10^?4 M) produced a dose-dependent simulation of the GTP-binding activity of G-proteins in plasma membrane fractions of the brain striatum and cardiac muscle in rats. The effect of peptide I was more pronounced and decreased to a considerable degree in the presence of the C-terminal 385–394 peptide of the G-protein α_s-subunit that selectively disrupts interaction of receptors with G_s-protein. Peptide I reduced markedly affinity of serotonin (agonist) to the serotonin striatum receptors, whereas peptide II inhibited to the significant extent the binding of dihydroalprenolol (antagonist) to β-adrenergic receptors in cardiac muscle. Peptide I, unlike peptide II, decreased essentially the high affinity binding of β-agonist isoproterenol. The obtained data indicate the ability of polycationic peptides to activate G₁-proteins, to disturb their coupling with receptor, and to affect binding properties of the receptor. There are differences in molecular mechanisms of action of peptides with different structures on G-proteins and receptors.