Wil J.A. Van Marrewijk

Apolipophorin II/I, apolipoprotein B, vitellogenin, and microsomal triglyceride transfer protein genes are derived from a common ancestor.

Abstract. Large lipid transfer proteins (LLTP) are nonexchangeable apolipoproteins and intracellular lipid-exchange proteins involved in the assembly, secretion, and metabolism of lipoproteins. We have identified contiguous conserved sequence motifs in alignments of insect apolipophorin II/I precursor (apoLp-II/I), human apolipoprotein B (apoB), invertebrate and vertebrate vitellogenins (VTG), and the large subunit of mammalian microsomal triglyceride transfer protein (MTP). Conserved motifs present in the N-terminal part of nonexchangeable apolipoproteins encompass almost completely the large subunit of MTP, suggesting a derivation from a common ancestral functional unit, termed large lipid transfer (LLT) module. Divergence of LLTP from a common ancestor is supported by (1) the statistical significance of the combined match scores obtained after motif-based database searches, (2) the presence of several identical amino acid residues in all LLTP sequences currently available, (3) the conservation of hydrophobic clusters in an α-helical domain, (4) the phylogenetic analysis of the conserved sequences related to the von Willebrand factor D (VWD) module identified in nonexchangeable apolipoproteins, and (5) the presence of four and one ancestral exon boundaries in the LLT and VWD modules, respectively. Our data indicate that the genes coding for apoLp-II/I, apoB, VTG, and the MTP large subunit are members of the same multigene superfamily. LLTP have emerged from an ancestral molecule designed to ensure a pivotal event in the intracellular and extracellular transfer of lipids and liposoluble substances.

Adipokinetic hormones of insect: Release, signal transduction, and responses

Flight activity of insects provides an attractive yet relatively simple model system for regulation of processes involved in energy metabolism. This is particularly highlighted during long-distance flight, for which the locust constitutes a well-accepted model insect. Peptide adipokinetic hormones (AKHs) are synthesized and stored by neurosecretory cells of the corpus cardiacum, a neuroendocrine gland connected with the insect brain. The actions of these hormones on their fat body target cells trigger a number of coordinated signal transduction processes which culminate in the mobilization of both carbohydrate (trehalose) and lipid (diacylglycerol). These substrates fulfill differential roles in energy metabolism of the contracting flight muscles. The molecular mechanism of diacylglycerol transport in insect blood involving a reversible conversion of lipoproteins (lipophorins) has revealed a novel concept for lipid transport in the circulatory system. In an integrative approach, recent advances are reviewed on the consecutive topics of biosynthesis, storage, and release of insect AKHs, AKH signal transduction mechanisms and metabolic responses in fat body cells, and the dynamics of reversible lipophorin conversions in the insect blood.

Alternative lipid mobilization: The insect shuttle system

Lipid mobilization in long-distance flying insects has revealed a novel concept for lipid transport in the circulatory system during exercise. Similar to energy generation for sustained locomotion in mammals, the work accomplished by non-stop flight activity is powered by oxidation of free fatty acids (FFA) derived from endogenous reserves of triacylglycerol. The transport form of the lipid, however, is diacylglycerol (DAG), which is delivered to the flight muscles associated with lipoproteins. In the insect system, the multifunctional lipoprotein, high-density lipophorin (HDLp) is loaded with DAG while additionally, multiple copies of the exchangeable apolipoprotein, apoLp-III, associate with the expanding particle. As a result, lipid-enriched low-density lipophorin (LDLp) is formed. At the flight muscles, LDLp-carried DAG is hydrolyzed and FFA are imported into the muscle cells for energy generation. The depletion of DAG from LDLp results in the recovery of both HDLp and apoLp-III, which are reutilized for another cycle of DAG transport. A receptor for HDLp, identified as a novel member of the vertebrate low-density lipoprotein (LDL) receptor family, does not seem to be involved in the lipophorin shuttle mechanism operative during flight activity. In addition, endocytosis of HDLp mediated by the insect receptor does not seem to follow the classical mammalian LDL pathway.Many structural elements of the lipid mobilization system in insects are similar to those in mammals. Domain structures of apoLp-I and apoLp-II, the non-exchangeable apolipoprotein components of HDLp, are related to apoB100. ApoLp-III is a bundle of five amphipathic α-helices that binds to a lipid surface very similar to the four-helix bundle of the N-terminal domain of human apoE. Despite these similarities, the functioning of the insect lipoprotein in energy transport during flight activity is intriguingly different, since the TAG-rich mammalian lipoproteins play no role as a carrier of mobilized lipids during exercise and besides, these lipoproteins are not functioning as a reusable shuttle for lipid transport. On the other hand, the deviant behavior of similar molecules in a different biological system may provide a useful alternative model for studying the molecular basis of processes related to human disorders and disease.

New insights into adipokinetic hormone signaling

Flight activity of insects comprises one of the most intense biochemical processes known in nature, and therefore provides an attractive model system to study the hormonal regulation of metabolism during physical exercise. In long-distance flying insects, such as the migratory locust, both carbohydrate and lipid reserves are utilized as fuels for sustained flight activity. The mobilization of these energy stores in Locusta migratoria is mediated by three structurally related adipokinetic hormones (AKHs), which are all capable of stimulating the release of both carbohydrates and lipids from the fat body. To exert their effects intracellularly, these hormones induce a variety of signal transduction events, involving the activation of AKH receptors, GTP-binding proteins, cyclic AMP, inositol phosphates and Ca2+. In this review, we discuss recent advances in the research into AKH signaling. This not only includes the effects of the three AKHs on each of the signaling molecules, but also crosstalk between signaling cascades and the degradation rates of the hormones in the hemolymph. On the basis of the observed differences between the three AKHs, we have tried to construct a physiological model for their action in locusts, in order to answer a fundamental question in endocrinology: why do several structurally and functionally related peptide hormones co-exist in locusts (and animals in general), when apparently one single hormone would be sufficient to exert the desired effects? We suggest that the success of the migratory locust in performing long-distance flights is in part based on this neuropeptide multiplicity, with AKH-I being the strongest lipid-mobilizing hormone, AKH-II the most powerful carbohydrate mobilizer and AKH-III, a modulatory entity that predominantly serves to provide the animal with energy at rest.

Molecular characterization and gene expression in the eye of the apolipophorin II/I precursor from Locusta migratoria

The transport of lipids via the circulatory system of animals constitutes a vital function that uses highly specialized lipoprotein complexes. In insects, a single lipoprotein, lipophorin, serves as a reusable shuttle for the transport of lipids between tissues. We have found that the two nonexchangeable apolipoproteins of lipophorin arise from a common precursor protein, apolipophorin II/I (apoLp‐II/I). To examine the mechanisms of transport of lipids and liposoluble substances inside the central nervous system, this report provides the molecular cloning of a cDNA encoding the locust apoLp‐II/I. We have recently shown that this precursor protein belongs to a superfamily of large lipid transfer proteins (Babin et al. [ 1999 ] J. Mol. Evol. 49:150–160). We determined that, in addition to its expression in the fat body, the locust apoLp‐II/I is also expressed in the brain. Part of the signal resulted from fat body tissue associated with the brain; however, apoLp‐II/I was strongly expressed and the corresponding protein detected, in pigmented glial cells of the lamina underlying the locust retina and in cells or cellular processes interspersed in the basement membrane. The latter finding strongly suggests an implication of apolipophorins in the transport of retinoids and/or fatty acids to the insect retina. J. Comp. Neurol. 427:546–558, 2000.

Adipokinetic hormone-induced influx of extracellular calcium into insect fat body cells is mediated through depletion of intracellular calcium stores

Adipokinetic hormone I (AKH I) needs extracellular Ca2+ for its activating action on glycogen phosphorylase in locust fat body in vitro. TMB-8 reduces this AKH effect significantly, indicating that for a major part, hormone action also requires the mobilization of Ca2+ from intracellular stores. Using 45Ca2+, AKH was shown to stimulate both the influx and the efflux of Ca2+. Thapsigargin also enhances the influx of extracellular Ca2+ into the fat body cells, indicating that the stimulating effect of AKH on Ca2+ influx may be mediated through depletion of intracellular Ca2+ stores as well. AKH is known to enhance cAMP levels in locust fat body. We show that elevation of cAMP with forskolin or theophylline leads to activation of glycogen phosphorylase, both in the presence and in the absence of extracellular Ca2+. The present data are discussed in an attempt to elucidate further the mechanism underlying transduction of the hormonal signal in locust fat body.

Differential induction of inositol phosphate metabolism by three adipokinetic hormones

Many (in)vertebrates simultaneously release several structurally and functionally related hormones; however, the relevance of this phenomenon is poorly understood. In the locust e.g. each of three adipokinetic hormones (AKHs) is capable of controlling mobilization of carbohydrate and lipid from fat body stores, but it is unclear why three AKHs coexist. We now demonstrate disparities in the signal transduction of these hormones. Massive doses of the AKHs stimulated total inositol phosphate (InsPn) production in the fat body biphasicly, but time courses were different. Inhibition of phospholipase C (PLC) resulted in attenuation of both InsPn synthesis and glycogen phosphorylase activation. The AKHs evoked differential formation of individual [3H]InsPn isomers (InsP(1-6)), the effect being most pronounced for InsP3. 40 nM of AKH-I and -III induced a substantial rise in total InsPn and [3H]InsP3 at short incubations, whereas the AKH-II effect was negligible. At a more physiological dose of 4 nM, the AKHs equally enhanced Ins(1,4,5)P3 levels. The InsP3 effect was most prolonged for AKH-III. These subtle differences in InsPn metabolism, together with earlier findings on differences between the AKHs, support the hypothesis that each AKH exerts specific biological functions in the overall syndrome of energy mobilization during flight.

Insect adipokinetic hormone stimulates inositol phosphate metabolism: roles for both Ins(1,4,5)P3 and Ins(1,3,4,5)P4 in signal transduction?

Adipokinetic hormones (AKHs) control the mobilization of energy reserves from the insect fat body as fuels for flight activity. As a part of our investigations on AKH signal transduction, we demonstrate in this study that the inositol lipid cycle may be involved in the action of AKH-I on fat body of the migratory locust. We show that [3H]inositol is incorporated into fat body phosphoinositides in vitro, whose hydrolysis leads to the formation of the following inositol phosphates (InsPs): Ins(1 and/or 3)P, Ins(4)P, Ins(1,3)P2, Ins(1,4)P2, Ins(3,4)P3, Ins(1,3,4)P3, Ins(1,4,5)P3 and Ins(1,3,4,5)P4. AKH stimulates the formation of these isomers, eliciting an increase in radioactivity of total InsPs already after 1 min. Mass measurements show that Ins(1,4,5)P3 levels are substantially enhanced by AKH, which is indicative of hormonal activation of phospholipase C. In cell-free tissue preparations, Ins(1,4,5)P3 is metabolized through dephosphorylation as well as further phosphorylation. Ins(1,3,4,5)P4 is dephosphorylated primarily to Ins(1,3,4)P3, although the ability for its reconversion to Ins(1,4,5)P3 suggests that in vivo Ins(1,3,4,5)P4 may function as a rapidly mobilizable pool for Ins(1,4,5)P3 generation. Metabolic pathways for the conversion of InsPs to inositol in the locust fat body are proposed.

Formation of partially phosphorylated glycogen phosphorylase in the fat body of the migratory locust

Abstract Glycogen phosphorylase b partially purified from fat body of migratory locusts was incubated with phosphorylase kinase and [γ- 32 P]ATP. DEAE-Sephacel chromatography revealed the conversion of phosphorylase b (absolute AMP dependence for activity) to almost equal amounts of phosphorylases ab (high AMP dependence) and a (largely AMP independent). Electrophoresis and autoradiography showed the incorporation of 32 P into the ab and a forms, and its absence from phosphorylase b . The labeling of phosphorylases ab and a , both being dimers of identical subunits, corresponded to respectively 1 and 2 mol phosphate incorporated/mol of enzyme. This suggests that in phosphorylase a both subunits are phosphorylated with one single phosphate group/subunit, while in phosphorylase ab only one subunit is phosphorylated.

The phospholipase C signaling pathway in locust fat body is activated via Gq and not affected by cAMP

Crosstalk between signal transduction pathways provides a complex intracellular avenue for fine tuning of hormone-induced signals. Over the last few years, we have studied the signaling mechanisms of three locust adipokinetic hormones (AKHs), which control mobilization of energy reserves from insect fat body as fuels for flight and transduce their signals via adenylyl cyclaseand phospholipase C- (PLC) dependent pathways. In this study, we examine possible crosstalk between these signaling routes. We show that cAMP does not affect basal and AKH-stimulated inositol phosphate (InsPn) production. Incubation of fat body with aluminium fluoride, an activator of G proteins, increased InsP n levels by 77%, whereas cholera toxin and pertussis toxin were ineffective. This implies that fat body PLC is not activated by Gbg, but possibly by Gqa. The involvement of this G protein in AKH signaling was demonstrated by our observation that the GPAntagonist-2A, which antagonizes Gq, attenuated glycogen phosphorylase activation by AKH-I. As plasma membrane Ca 2 + channels may constitute another target for cAMP-mediated modulation, we studied the type of channels involved in AKH signaling using a variety of L-, N- and T-type Ca 2 + channel inhibitors. None of these blocked AKH-induced glycogen phosphorylase activation, suggesting that voltage-dependent Ca 2 + channels do not mediate AKH-induced Ca 2 + influx.