The structurally similar neuropeptides adipokinetic hormone I and II are derived from similar, very small mRNAs

The grasshopper neuropeptides adipokinetic hormone (AKH) I and II were among the first of an extensive family of structurally similar arthropod hormones and neuroregulators to be isolated and sequenced. This paper reports the cloning of cDNAs derived from the unusually small mRNAs (550 bases) which code for the precursors of AKH I and II from Schistocerca nitans. Sequence analysis of the cDNAs indicates that AKH I and II are derived from small precursor proteins (63 and 61 amino acids) which are 55% identical in amino acid sequence. Each contains a 22-amino acid hydrophobic leader sequence followed by the AKH I or II sequence and an additional 28-amino acid carboxyl-terminal peptide of unknown function. Significant homology at the nucleic acid level (64% identity) is confined to the coding region of the mRNA sequences. Preliminary DNA blot analyses suggest that a single gene codes for each, and that the genes for AKH I and II may be linked. Genomic blots from various tissues fail to suggest that the high level of expression of AKH in the corpora cardiaca is due to tissue specific gene amplification.     

In silico cloning of genes encoding neuropeptides, neurohormones and their putative G-protein coupled receptors in a spider mite

The genome of the spider mite was prospected for the presence of genes coding neuropeptides, neurohormones and their putative G-protein coupled receptors. Fifty one candidate genes were found to encode neuropeptides or neurohormones. These include all known insect neuropeptides and neurohormones, with the exception of sulfakinin, corazonin, neuroparsin and PTTH. True orthologs of adipokinetic hormone (AKH) were neither found, but there are three genes encoding peptides similar in structure to both AKH and the AKH-corazonin-related peptide. We were also unable to identify the precursors for pigment dispersing factor (PDF) or the recently discovered trissin. However, the spider mite probably does have such genes, as we found their putative receptors. A novel arthropod neuropeptide gene was identified that shows similarity to previously described molluscan neuropeptide genes and was called EFLamide. A total of 65 putative neuropeptide GPCR genes were also identified, of these 58 belong to the A-family and 7 to the B-family. Phylogenetic analysis showed that 50 of them are closely related to insect GPCRs, which allowed the identification of their putative ligand in 39 cases with varying degrees of certainty. Other spider mite GPCRs however have no identifiable orthologs in the genomes of the four holometabolous insect species best analyzed. Whereas some of the latter have orthologs in hemimetabolous insect species, crustaceans or ticks, for others such arthropod homologs are currently unknown.     

New insights in Adipokinetic Hormone (AKH) precursor processing in Locusta migratoria obtained by capillary liquid chromatography-tandem mass spectrometry

After translation, the AKH I and AKH II precursors form three dimeric constructs prior to further processing into the respective AKHs and three dimeric Adipokinetic Hormone Precursor Related Peptides or APRPs (two homodimers and one heterodimer).By capillary liquid chromatography-tandem mass spectrometry we demonstrate that the APRPs in Locusta migratoria are further processed to form two smaller neuropeptides: DAADFADPYSFL (residue 36 to 47 of the AKH I precursor) and YADPNADPMAFL (residue 34 to 45 of the AKH II precursor). The peptides are designated as Adipokinetic Hormone Joining Peptide 1 (AKH-JP I) and 2 (AKH-JP II) respectively. Within the AKH I and AKH II precursor molecules, the classic KK and RR processing sites separate the AKH-JPs from the AKH I and II respectively. At the carboxyterminus, both AKH-JP I and II are flanked by Tyr-Arg, a cleaving site not described before. Such an unusual cleavage site suggests the presence, in the corpora cardiaca, of specific convertases. The AKH-JP-II does not stimulate lipid release from the fat body nor does it stimulate glycogen phosphorylase activity, both key functions of AKH.     

Identification and Initial Characterization of Adipokinetic Hormone-Like Immunoreactive Peptides of Rat Origin

An antiserum was raised to adipokinetic hormone (AKH), a 10-amino-acid-residue peptide found in the arthropod Locusta migratoria. The antiserum demonstrated not only immunocytochemical reaction with some other arthropod species, but also stained many areas of the rat CNS, certain islet cells of the pancreas, and some anterior pituitary cells. The pattern of staining was unlike that for any known rat neuropeptide or hormone. With the antiserum used as the detection system, HPLC and high-voltage electrophoresis yielded two peptides that were purified to homogeneity from rat hypothalamic median eminence. These peptides have unique amino acid compositions, indicating they may be heretofore unknown rat neuropeptides.     

Invertebrate neuropeptide hormones

The development of a long-term research program on the neurosecretory hormones of arthropods is described. The purification and full characterization of the first invertebrate neurohormones, the red pigment-concentrating hormone (RPCH) and the distal retinal pigment hormone (DRPH) demonstrated that they are peptides, an octapeptide and an octadecapeptide, respectively. Physiological function studies with the pure hormones and their synthetic preparations showed that the RPCH acts as a general pigment-concentrating hormone (PCH), and that the DRPH, in addition to its light-adaptive function, also constitutes a general pigment-dispersing hormone (PDH). In the regulation of the color-adaptation of the animals, the two hormones act as antagonists. The chromatophorotropic activities are widely distributed within the arthropod neuroendocrine systems. Purification of the pigment-concentrating activities from the locust corpora cardiaca lead to the isolation and characterization of the first insect neurohormones, the adipokinetic hormones (AKH I and AKH II). These two hormones, AKH I being a decapeptide and AKH II being an octapeptide, are close structural analogs to the crustacean PCH, demonstrating a common evolution of arthropod neurohormones. The hormones of this PCH-family all cross-react, but structure-function studies of the hormones show that quite different parts of their structure are involved in their binding to the various receptors.     

Chemical Properties and Physiological Actions of Crustacean Chromatophorotropins

The crustacean pigment-translocating hormones, the red pigment-concentratinghormone (RPCH), an octapeptide, and the light-adapting distalretinal pigment hormone (DRPH), an octadecapeptide, are thefirst invertebrate neurohormones to be fully characterized.Studies with both purified and synthetic hormones show that,in certain decapods, RPCH is a general pigment-concentratinghormone (PCH), affecting the pigments of all kinds of chromatophores(erythrophores, xanthophores, leucophores and melanophores);the DRPH seems to serve not only light-adapting function, butalso act as a general chromatophore pigment-dispersing hormone(PDH). The two hormones thus function as antagonists when regulatingthe color-adaptation of the decapod crustaceans. PCH activityis widely distributed within the arthropod endocrine systems.The first characterized insect neurohormones, the locust adipokinetichormones (AKH I and AKH II), show close structural similaritiesto the crustacean hormone, indicating a common evolution ofsome of the arthropod neurohormones. Physiological studies ofthe three hormones (RPCH, AKH I, and AKH II) and their syntheticanalogs show that they crossreact, i.e., they all exhibit pigment-concentratingactivity when tested on decapod crustaceans, adipokinetic activitywhen tested on locusts, and hyperglycemic activity when testedon cockroaches, although each of the hormones is more potentin its own system. Structure-function studies show, however,that quite different binding-site requirements exist for thehormones in activating their receptors on the various targettissues. The physiological specificity in their action thereforeseems to depend on a differential evolution of the hormone receptors.     

Cross-phyletic bioactivity of arthropod neurohormones and molluscan ganglion extracts: evidence of an extended peptide family

Two structurally related arthropod neuropeptides, red pigment concentrating hormone (RPCH) and adipokinetic hormone (AKH), are potent excitors of the heart of the clam Mercenaria mercenaria. The response is bimodal: whereas the threshold for affected hearts is 1-3 X 10(-9) M, about 40% of the preparations are virtually unresponsive. Aqueous extracts of Mercenaria ganglia contain a substance which concentrates the red pigment in the erythrophores of intact destalked Uca pugilator and even of its isolated legs. This substance is retained on Sephadex G-15 and co-elutes with synthetic shrimp RPCH. The active fractions also concentrate the erythrophores and the leucophores of destalked shrimp (Penaeus). Neither dopamine nor the molluscan neuropeptide FMRFamide had any chromatophorotropic effect in these assays. The activity of the ganglion extracts was abolished by digestion with chymotrypsin. In conclusion, molluscan ganglion extracts contain a peptide factor, possibly an analog of RPCH, that concentrates the pigments of crustacean chromatophores by a direct action on the cells.     

Vanessa cardui adipokinetic hormone (Vanca-AKH) in butterflies and a moth

Small neuropeptides of the adipokinetic hormone/red pigment-concentrating hormone (AKH/RPCH) family regulate energy metabolism in insects. Within lepidopterans, the nonapeptide Manduca sexta AKH (Manse-AKH) represents a widely occurring AKH, whereas the decapeptide Helze-HrTH (at first isolated from Heliothis zea) seems to be restricted to moths. Here we show that Vanca-AKH, a non-amidated undecapeptide which we recently found in the painted lady butterfly, Vanessa cardui, is also present in the retrocerebral complex of several other butterflies (Danaus plexippus, Precis coenia, Aglais urticae) and a moth (Spodoptera frugiperda). This study also demonstrates the power of modern nano-electrospray-quadrupole TOF tandem mass spectrometry in the sequence confirmation of peptides from minute amounts of small neuropeptides.     

A phylogenetic analysis of the adipokinetic neuropeptides of Ensifera

Abstract.  Adipokinetic neuropeptides, from the corpora cardiaca of various species of the suborder Ensifera, encompassing members of all superfamilies (except the Gryllacridoidea), were isolated by liquid chromatography, and identified structurally by comparison of retention times and mass spectrometry data with respect to information from known members of this peptide family. Ensiferan species always contain only one adipokinetic hormone (AKH) peptide, as assessed for a few species by monitoring typical AKH mass peaks from a crude corpora cardiaca extract. This AKH is an octapeptide, and is either Scg-AKH-II (pGlu-Leu-Asn-Phe-Ser-Thr-Gly-Trp amide) which occurs in all Tettigoniidea (except Schizodactyloidea) and in Gryllotalpoidea, or Grb-AKH (pGlu-Val-Asn-Phe-Ser-Thr-Gly-Trp amide) which occurs in Grylloidea (except Gryllotalpoidea) and Schizodactyloidea. Using the structural information of these neuropeptides in conjunction with morpho-anatomical characters, these data are interpreted in a phylogenetic framework. The lack of a decapeptide and the presence of the octapeptide Scg-AKH-II are ancestral in Ensifera. The ancestral Scg-AKH-II twice underwent an independent and convergent modification to Grb-AKH.     

The fruitfly Drosophila melanogaster contains a novel charged adipokinetic-hormone-family peptide.

A member of the RPCH/AKH (red-pigment-concentrating hormone/adipokinetic hormone) family of arthropod neuropeptides was identified in the fruitfly Drosophila melanogaster, and its structure was determined by automated Edman degradation and m.s. using fast-atom-bombardment ionization and a tandem hybrid instrument capable of high sensitivity. The sequence of this peptide, which we call 'DAKH', is pGlu-Leu-Thr-Phe-Ser-Pro-Asp-Trp-NH2 (where pGlu is pyroglutamic acid and Trp-NH2 is tryptophan carboxyamide). H.p.l.c. analyses of extracts of the three body segments revealed that more than 80% of the peptide is contained in the thorax. Although DAKH is typical of family members in its general structure and distribution in the animal, it is unique in containing a residue which is charged under physiological conditions. The evolutionary significance of this change is considered.     

Melatonin-induced neuropeptide release from isolated locust corpora cardiaca

A method, based on a combination of mass spectrometry and liquid chromatography, was developed to investigate the release of neuropeptides from isolated locust corpora cardiaca. Melatonin, octopamine, trehalose and forskolin were administered to the perifused glands. The neuropeptides present in the releasates (spontaneous versus induced) were visualized by either conventional or capillary HPLC. Identification was achieved by means of MALDI-TOF MS and/or nanoflow-LC-Q-TOF MS. The observed effects of these chemicals regarding AKH release were in line with previous studies and validate the method. The most important finding of this study was that administration of melatonin stimulated the release of adipokinetic hormone precursor related peptides (APRP 1 and APRP 2), neuroparsins (NP A1, NP A2 and NP B) and diuretic peptide.     

An invertebrate [hydroxyproline]-modified neuropeptide: further evidence for a close evolutionary relationship between insect adipokinetic hormone and mammalian gonadotropin hormone family

An octapeptide of the adipokinetic hormone (AKH) peptide family is identified in the corpora cardiaca of the stink bug, Nezara viridula, by ESI-MS^N (electrospray ionization multistage MS). This is the second AKH in N. viridula and it has a hydroxyproline residue at position 6, whereas the major AKH (known as Panbo-RPCH) has Pro as the sixth amino acid residue. The correct sequence assignment of [Hyp⁶]-Panbo-RPCH is confirmed by retention time and MS spectra of the synthetic peptide. Various extraction procedures were followed to ascertain whether the hydroxylation is an artefact of extraction, or whether it is due to a true post-translational modification at the prohormone level. The proline hydroxylation is unique for invertebrate neuropeptides, while it has been described in the vertebrate gonadotropin-releasing hormone (GnRH). The current finding is another piece of evidence that AKH and GnRH form a peptide superfamily and are closely related evolutionarily. Biologically, [Hyp⁶]-Panbo-RPCH is active in vivo as an AKH, causing hyperlipaemia in the stink bug at low doses, indicating again that it is an endogenous, mature and functional hormone in this insect species.     

Metabolism of Stored Reserves in Insect Fat Body: Hormonal Signal Transduction Implicated in Glycogen Mobilization and Biosynthesis of the Lipophorin System*

The mobilization of carbohydrate and lipid reserves from the insect fat body as fuels for migratory flight activity is controlled by adipokinetic hormone (AKH), of which in Locusta migratoria three different forms occur: AKH-I, -II and -III. In fat body in vitro, each AKH is capable of activating glycogen phosphorylase and of stimulating cAMP production, but only in the presence of extracellular Ca²⁺. The hormones stimulate both the influx and the efflux of Ca²⁺, the higher influx probably causing an increase in intracellular [Ca²⁺]. AKH enhances the production of inositol phosphates among which inositol 1,4,5-triphosphate may mediate the mobilization of Ca²⁺ from intracellular stores. Evidence is presented in favor of the occurrence of a capacitative calcium entry mechanism. Results suggest that transduction of the AKH signal occurs through stimulatory G protein-coupled receptor(s). A tentative model is presented for the interactions between the AKH signaling pathways in the locust fat body cell. AKH-induced lipid mobilization during flight requires the presence in the insect blood of high-density lipophorin (HDLp) particles and apolipophorin III (apoLp-III). Both protein components are synthesized in the fat body. In the locust, the two integral, nonexchangeable HDLp apolipophorins (apoLp-I and -II) were shown to originate from a common precursor; an mRNA of 10.3 kb seems to code for this precursor protein. The models proposed for lipophorin assembly and secretion in a number of insects are not in agreement. The exchangeable apoLp-III may occur in two or more isoforms; locust apoLp-III is secreted from the fat body as one of the two isoforms and in the hemolymph converted into the truncated second one. The rationale for this process is as yet unknown.     

Cloning and characterization of the adipokinetic hormone receptor from the cockroach Periplaneta americana

Cockroaches have long been used as insect models to investigate the actions of biologically active neuropeptides. Here, we describe the cloning and functional expression in Chinese hamster ovary cells of an adipokinetic hormone (AKH) G protein-coupled receptor from the cockroach Periplaneta americana. This receptor is only activated by various insect AKHs (we tested eight) and not by a library of 29 other insect or invertebrate neuropeptides and nine biogenic amines. Periplaneta has two intrinsic AKHs, Pea-AKH-1, and Pea-AKH-2. The Periplaneta AKH receptor is activated by low concentrations of both Pea-AKH-1 (EC50, 5 x 10(-9)M), and Pea-AKH-2 (EC50, 2 x 10(-9)M). Insects can be subdivided into two evolutionary lineages, holometabola (insects with a complete metamorphosis during development) and hemimetabola (incomplete metamorphosis). This paper describes the first AKH receptor from a hemimetabolous insect.     

Structures of two cockroach neuropeptides assigned by fast atom bombardment mass spectrometry

Amino acid sequences have been assigned to two cockroach neuropeptides (Glu-Val-Asn-Phe-Ser-Pro-Asn-Trp-NH₂, M I, and Glu-Leu-Thr-Phe-Thr-Pro-Asn-Trp-NH₂, M II) by application of fast atom bombardment mass spectrometry, including high resolution and linked scan (metastable) studies. The peptides show considerable homology with two other invertebrate neuropeptides, adipokinetic hormone (AKH, from a locust) and red pigment concentrating hormone (RPCH, from a prawn), whose fast atom bombardment spectra were also studied. M I and M II are thus members of a family of structurally-related invertebrate neuropeptides.     

Dimer structure of a neuropeptide precursor established: consequences for processing

A prohormone (P1) of locust adipokinetic hormone I (AKH I) is shown here to be a homodimer of a 41 residue subunit called the A-chain. The A-chain, from the N terminal, consists of AKH I (10 amino acids starting with pyroglutamate) followed by a Gly-Lys-Arg processing site and then a 28 residues called the alpha chain containing a single cysteine and a potential Arg-Lys processing site. When processed each molecule of the homodimer precursor yields two copies of AKH I and one alpha chain homodimer. We call the alpha-alpha homodimer product of P1 processing AKH precursor related peptide 1 or APRP 1. The Arg-Lys dibasic pair found within the alpha chain is not cleaved in vivo. Our results show that neuropeptide precursors can be dimers and that dimer products can be synthesized by processing of a preformed dimer precursor rather than by dimerization of independent subunits.     

Adipokinetic hormone counteracts oxidative stress elicited in insects by hydrogen peroxide: in vivo and in vitro study

The role of adipokinetic hormone (AKH) in counteracting oxidative stress elicited in the insect body is studied in response to exogenously applied hydrogen peroxide, an important metabolite of oxidative processes. In vivo experiments reveal that the injection of hydrogen peroxide (8 µmol) into the haemocoel of the firebug, Pyrrhocoris apterus L. (Heteroptera: Pyrrhocoridae) increases the level of AKH by 2.8‐fold in the central nervous system (CNS) and by 3.8‐fold in the haemolymph. The injection of hydrogen peroxide also increases the mortality of experimental insects, whereas co‐injection of hydrogen peroxide with Pyrap‐AKH (40 pmol) reduces mortality to almost control levels. Importantly, an increase in haemolymph protein carbonyl levels (i.e. an oxidative stress biomarker) elicited by hydrogen peroxide is decreased by 3.6‐fold to control levels when hydrogen peroxide is co‐injected with Pyrap‐AKH. Similar results are obtained using in vitro experiments. Oxidative stress biomarkers such as malondialdehyde and protein carbonyls are significantly enhanced upon exposure of the isolated CNS to hydrogen peroxide in vitro, whereas co‐treatment of the CNS with hydrogen peroxide and Pyrap‐AKH reduces levels significantly. Moreover, a marked decrease in catalase activity compared with controls is recorded when the CNS is incubated with hydrogen peroxide. Incubation of the CNS with hydrogen peroxide and Pyrap‐AKH together curbs the negative effect on catalase activity. Taken together, the results of the present study provide strong support for the recently published data on the feedback regulation between oxidative stressors and AKH action, and implicate AKH in counteracting oxidative stress. The in vitro experiments should facilitate research on the mode of action of AKH in relation to oxidative stress, and could help clarify the key pathways involved in this process.     

Neuropeptides modulate the transmitter-induced glycogenolysis and gluconeogenesis in leech segmental ganglia

The effects of four neuropeptides (AKH, FMRFamide, proctolin, VIP) on the alterations in glycogen levels induced by other transmitters were studied in isolated leech segmental ganglia. With the exception of FMRFamide, which produced a small increase (14%) in glycogen, the peptides were by themselves without effect on the glycogen levels. Proctolin abolished the glycogenolytic effects of 5-HT, dopamine and octopamine but not histamine. AKH in combination with ACh produced glycogenolysis although each by themselves were ineffective. AKH modified the effects of other transmitters in different ways i.e. by reduction or reversal of effect. VIP and noradrenaline produced an increase in glycogen (cf. noradrenaline alone which decreased glycogen), but did not modify the effects of the other transmitters. FMRFamide produced a complex variety of modulatory effects on the other transmitters. It is concluded that the glycogen stores in leech ganglia, which are localized principally in the glial cells, may be controlled in complex ways by the different combinations of monoamines, amino acids and neuropeptides.