Endogenous Adipokinetic Hormone Research Articles

In Silico Screening for Pesticide Candidates against the Desert Locust Schistocerca gregaria.

Adipokinetic hormone (AKH) is one of the most important metabolic neuropeptides in insects, with actions similar to glucagon in vertebrates. AKH regulates carbohydrate and fat metabolism by mobilizing trehalose and diacylglycerol into circulation from glycogen and triacylglycerol stores, respectively, in the fat body. The short peptide (8 to 10 amino acids long) exerts its function by binding to a rhodopsin-like G protein-coupled receptor located in the cell membrane of the fat body. The AKH receptor (AKHR) is, thus, a potential target for the development of novel specific (peptide) mimetics to control pest insects, such as locusts, which are feared for their prolific breeding, swarm-forming behavior and voracious appetite. Previously, we proposed a model of the interaction between the three endogenous AKHs of the desert locust, Schistocerca gregaria, and the cognate AKHR (Jackson et al., Peer J. 7, e7514, 2019). In the current study we have performed in silico screening of two databases (NCI Open 2012 library and Zinc20) to identify compounds which may fit the endogenous Schgr-AKH-II binding site on the AKHR of S. gregaria. In all, 354 compounds were found to fit the binding site with glide scores < −8. Using the glide scores and binding energies, 7 docked compounds were selected for molecular dynamic simulation in a phosphatidylcholine membrane. Of these 7 compounds, 4 had binding energies which would allow them to compete with Schgr-AKH-II for the receptor binding site and so are proposed as agonistic ligand candidates. One of the ligands, ZINC000257251537, was tested in a homospecific in vivo biological assay and found to have significant antagonistic activity.

Structure-Activity Studies on the Hypertrehalosemic Hormone II of the Stick Insect Carausius morosus (Phasmatodea): Carbohydrate-Mobilization and Cardio-Stimulatory Activities.

The corpora cardiaca of the Indian stick insect, Carausius morosus, synthesize two decapeptide neuropeptides of the adipokinetic hormone (AKH) family, both of which can increase the trehalose levels in the hemolymph when the stick insect is ligated between the head and the thorax. Here, we use two biological assays to assess the potencies of 19 AKH analogs in ligated C. morosus: the carbohydrate-mobilizing assay measures the change in the levels of circulating carbohydrates following injection of a substance, while the semi-exposed heart assay measures a change in heart beat rate after the peptide is applied onto the heart. With the endogenous AKH (Carmo-HrTH-II) as lead peptide, we report here on seven naturally-occurring AKH peptides (bioanalogs) selected for testing because of a single or double amino acid replacement, or for being octapeptides. Single amino acid substitutions by an alanine residue at all positions of Carmo-HrTH-II, as well as analogs modified at the termini were also investigated to give a comprehensive view of ligand-receptor interaction at the physiological level in a hemimetabolous insect that practices thanatosis (feigning death). Only small changes are elicited in the bioassays, but the results from the two tests are comparable bar one or two anomalies. Results show that analogs modified at the termini have no or reduced activity. Regarding structural requirements of a ligand, the C. morosus AKH receptor appears to be strict: octapeptides are not preferred and many of the decapeptide analogs failed to reach 50% activity relative to Carmo-HrTH-II. The data implies that the AKH receptor in C. morosus mostly does not tolerate shorter peptides and single amino acid replacements in most places of the native AKH peptide. This information is important if environmentally friendly insect-specific pesticides are made based on an insect AKH as lead peptide: stick insects that are normally not viewed as pest insects may not be easily targeted by cross-reactive AKH mimetics directed at harmful insects, due to the very specific amino acid requirements to activate the C. morosus AKH receptor.

The adipokinetic hormones and their cognate receptor from the desert locust, Schistocerca gregaria: solution structure of endogenous peptides and models of their binding to the receptor.

BackgroundNeuropeptides exert their activity through binding to G protein-coupled receptors (GPCRs). GPCRs are well-known drug targets in the pharmaceutical industry and are currently discussed as targets to control pest insects. Here, we investigate the neuropeptide adipokinetic hormone (AKH) system of the desert locust Schistocerca gregaria. The desert locust is known for its high reproduction, and for forming devastating swarms consisting of billions of individual insects. It is also known that S. gregaria produces three different AKHs as ligands but has only one AKH receptor (AKHR). The AKH system is known to be essential for metabolic regulation, which is necessary for reproduction and flight activity.MethodsNuclear magnetic resonance techniques (NMR) in a dodecylphosphocholin (DPC) micelle solution were used to determine the structure of the three AKHs. The primary sequence of the S. gregaria AKHR was used to construct a 3D molecular model. Next, the three AKHs were individually docked to the receptor, and dynamic simulation of the whole ligand–receptor complex in a model membrane was performed.ResultsAlthough the three endogenous AKHs of S. gregaria have quite different amino acids sequences and chain length (two octa- and one decapeptide), NMR experiments assigned a turn structure in DPC micelle solution for all. The GPCR-ModSim program identified human kappa opioid receptor to be the best template after which the S. gregaria AKHR was modeled. All three AKHs were found to have the same binding site on this receptor, interact with similar residues of the receptor and have comparable binding constants. Molecular switches were also identified; the movement of the receptor could be visually shown when ligands (AKHs) were docked and the receptor was activated.ConclusionsThe study proposes a model of binding of the three endogenous ligands to the one existing AKHR in the desert locust and paves the way to use such a model for the design of peptide analogs and finally, peptide mimetics, in the search for novel species-specific insecticides based on receptor–ligand interaction.

Analysis of Peptide Ligand Specificity of Different Insect Adipokinetic Hormone Receptors.

Adipokinetic hormone (AKH) is a highly researched insect neuropeptide that induces the mobilization of carbohydrates and lipids from the fat body at times of high physical activity, such as flight and locomotion. As a naturally occurring ligand, AKH has undergone quite a number of amino acid changes throughout evolution, and in some insect species multiple AKHs are present. AKH acts by binding to a rhodopsin-like G protein-coupled receptor, which is related to the vertebrate gonadotropin-releasing hormone receptors. In the current study, we have cloned AKH receptors (AKHRs) from seven different species, covering a wide phylogenetic range of insect orders: the fruit fly, Drosophila melanogaster, and the yellow fever mosquito, Aedes aegypti (Diptera); the red flour beetle, Tribolium castaneum, and the large pine weevil, Hylobius abietis (Coleoptera); the honeybee, Apis mellifera (Hymenoptera); the pea aphid, Acyrthosiphon pisum (Hemiptera); and the desert locust, Schistocerca gregaria (Orthoptera). The agonistic activity of different insect AKHs, including the respective endogenous AKHs, at these receptors was tested with a bioluminescence-based assay in Chinese hamster ovary cells. All receptors were activated by their endogenous ligand in the nanomolar range. Based on our data, we can refute the previously formulated hypothesis that a functional AKH signaling system is absent in the beneficial species, Apis mellifera. Furthermore, our data also suggest that some of the investigated AKH receptors, such as the mosquito AKHR, are more selective for the endogenous (conspecific) ligand, while others, such as the locust AKHR, are more promiscuous and can be activated by AKHs from many other insects. This information will be of high importance when further analyzing the potential use of AKHRs as targets for developing novel pest control agents.

Identification of the adipokinetic hormone of the large milkweed bugOncopeltus fasciatus

The adipokinetic hormone (AKH) of the large milkweed bug Oncopeltus fasciatus is isolated from an acidified methanolic extract of 200 corpora cardiaca, purified by single step reversed phase high-performance liquid chromatography (HPLC) and N-terminally deblocked using pyroglutamate aminopeptidase. The sequence is identified by Edman degradation and matrix assisted laser desorption/ionization-time of flight mass spectroscopy as pGlu-Leu-Asn-Phe-Ser-Pro-Asn-Trp amide. This structure is confirmed by chemical synthesis and coelution of native and synthetic peptide on HPLC. The AKH of O. fasciatus is identical to Tenmo-HrTH, a member of the adipokinetic/red pigment-concentrating hormone peptide family that had been isolated earlier from several tenebrionid beetles. Tenmo-HrTH causes a significant rise in the concentration of haemolymph lipids when injected into adult male and female O. fasciatus, but displays no hyperglycaemic activity. There is no indication of the presence of other AKHs in O. fasciatus. The large milkweed bug represents the first member of the seed bugs (Lygaeidae) for which the endogenous AKH has been identified.

Flight fuel and neuropeptidergic control of fuel mobilisation in the twig wilter, Holopterna alata (Hemiptera, Coreidae)

The corpus cardiacum of the twig wilter Holopterna alata contains a factor that elicits increases in the concentration of lipids in the haemolymph of twig wilters and migratory locusts and causes hypertrehalosaemia in American cockroaches. A hyperlipaemic neuropeptide was isolated from corpora cardiaca of H. alata in a single high-performance liquid chromatography step. The primary sequence of this peptide was assigned by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry, biological assay and co-elution with the synthetic peptide. The adipokinetic peptide of H. alata is an octapeptide with the sequence pGlu–Leu–Asn–Phe–Ser–Thr–Gly–Trp amide denoted Schgr–AKH-II which was sequenced previously from the corpora cardiaca of a number of Caelifera, Ensifera and some Hymenoptera. A dose of 1 pmol of synthetic Schgr–AKH-II causes a pronounced hyperlipaemic effect in the twig wilter. Physiological experiments with the twig wilter reveal that during flight periods of 3 min, the normally low carbohydrate concentration in the haemolymph is significantly diminished, whereas the lipid concentration stays constant in most cases. During a subsequent rest period of 60 min after a 3 min flight episode, however, the concentration of lipids in the haemolymph increases substantially and significantly, indicating that lipids, too, are a major fuel during flight of twig wilters. This is corroborated by the activation of the enzyme triacylglycerol (TAG) lipase in the fat body, but not in the flight muscles, by injection of 5 pmol of synthetic Schgr–AKH-II, the endogenous adipokinetic hormone that is thought to be released during flight. Moreover, in the thorax there is a significant decrease in the concentration of glycogen and lipids measured after flight plus 60 min of rest compared to non-flown twig wilters, whereas no significant changes were monitored for these substrates stored in the abdomen. When the change in lipid class composition was analysed during flight plus 60 min of rest, TAG which comprised the major class in all compartments analysed (thorax, abdomen, haemolymph) was significantly reduced in abdomen and thorax, and diacylglycerol was significantly increased in all three compartments. From all the data collected, it is concluded that lipids are the major fuel class for flight in H. alata and that the contribution of carbohydrates is minimal.

Flight or fight—the need for adipokinetic hormones

An attempt is made to show similarities in function and mode of action of mammalian adrenaline and the adipokinetic hormone (AKH) of insects. As specific case studies, the elucidation of primary structures of AKHs of heteropteran Hemiptera are presented and their function in flight metabolism in this taxon of insects is discussed in detail. All investigated species, representing the families Pentatomidae, Coreidae and Notonectidae, apparently rely (mainly) on lipid oxidation to provide energy for flight and, where measured, for swimming as well. In another case study, beetles which use proline as a fuel for flight, e.g., Pachnoda sinuata, need to oxidize fatty acids in order to produce acetyl CoA which will combine with alanine to resynthesise proline. Recent studies demonstrate that the injection of the endogenous AKH of P. sinuata and flight (upon which AKH is released) are able to activate the enzyme triacylglycerol lipase. The second messengers in this process are cAMP and Ca 2+.

Induced hyperlipaemia and immune challenge in locusts

Injections of immunogens, such as β-1,3-glucan or lipopolysaccharide (LPS), bring about a marked hyperlipaemia with associated changes in lipophorins and apolipophorin-III in the haemolymph of Locusta migratoria. These changes are similar to those observed after injection of adipokinetic hormone (AKH). The possibility that endogenous AKH is released as part of the response to these immunogens is investigated using passive immunisation against AKH-I, and measurement of AKH-I titre in the haemolymph after injection of immunogens. The data presented show that, despite the similarity of the changes brought about by the presence of immunogens in the haemolymph to those brought about by AKH, there is no release of endogenous AKH after injection of laminarin or LPS. A direct effect of the immunogens on release of neutral lipids by the fat body cannot be demonstrated in vitro, and the mechanism by which hyperlipaemia is induced during immune challenge remains uncertain.

Mode of action of neuropeptides from the adipokinetic hormone family

Neuropeptides of the adipokinetic hormone (AKH) family regulate inter alia mobilisation of various substrates from stores in the fat body of insects during episodes of flight. How is this achieved? In insects which exclusively oxidise carbohydrates for flight (cockroaches), or which oxidise carbohydrates in conjunction with lipids (locusts) or proline (a number of beetles), the endogenous AKHs bind to a G q-protein-coupled receptor, activate a phospholipase C and the resulting inositol trisphosphate releases Ca 2+ from internal stores. In addition, influx of extracellular Ca 2+ is increased and, via a kinase cascade, glycogen phosphorylase is activated, glucose-1-phosphate produced, and transformed to trehalose, which is released into the haemolymph. In locusts, additionally, adenylate cyclase is activated and cyclic AMP is synthesised. In insects which use lipids for sustained flight (locust, tobacco hornworm moth) or proline for flight (certain beetles), adenylate cyclase is activated after the AKHs bind to their respective G s-protein-coupled receptor. The resulting cyclic AMP, together with the messengers intra- and extracellular Ca 2+, activate a triacylglycerol lipase, which results in the production of 1,2 diacylglycerols (in locusts, moths) or (hypothetically) free fatty acids (fruit beetle).