Adipokinetic and Hypertrehalosemic Neurohormones

The adipokinetic hormones and hypertrehalosemic hormones of insects comprise a family of peptide hormones that primarily regulate the levels of energy metabolites, such as trehalose, diacylglycerol and proline that circulate in the hemolymph. These peptide hormones are products of neurosecretory neurons located in the corpora cardiaca, neuroendocrine glands attached to the brain. The structural organization of the insect corpora cardiaca is similar to the hypothalamus-neurohypophysis of the vertebrate endocrine system.

Historical Perspective

The existence of hypertrehalosemic hormones was discovered with the observation that injections of extracts of corpora cardiaca elevated the concentration of trehalose in the hemolymph of cockroaches (hypertrehalosemia). Unlike vertebrates that use glucose as the major blood carbohydrate, the hemolymph of insects generally contains the disaccharide trehalose, an α-1-1gluco-glucoside, as its major circulating carbohydrate. In addition, the enzyme glycogen phosphorylase in the fat body of cockroaches was demonstrated to be activated when these insects were injected with an extract from the corpora cardiaca. Subsequently, studies in locusts showed that injections of corpora cardiaca extracts elevated hemolymph diacylglycerols, instead of trehalose, and this action was referred to as an adipokinetic or hyperlipemic effect. Injections of locust corpora cardiaca extracts into cockroaches produced the hypertrehalosemic response, and vice versa. Hence, it was likely that the adipokinetic hormone (AKH) of locusts and the hypertrehalosemic hormone (HrTH) of cockroaches were related, or identical, peptides.

The locust adipokinetic hormone was isolated and characterized first. It was obtained from the migratory locust, Locusta migratoria, and its primary structure consisted of ten amino acids. It was designated Locmi-AKH-I according to the newest nomenclature for naming insect neurohormones. The amino acid composition and sequence of Locmi-AKH-I had a remarkable similarity to a previously reported red pigment-concentrating hormone (Panbo-RPCH) obtained from the shrimp Pandalus borealis and later found in various crustaceans. It was shown that Locmi-AKH-I was also present in the desert locust, Schistocerca gregaria.

Subsequently, both L. migratoria and S. gregaria were shown to contain a second adipokinetic hormone (Locmi-AKH-II and Schgr-AKH-II, respectively) that differed from each other by the amino acids in position 6. The two locust AKH-IIs were octapeptides with sequences similar to those of Locmi-AKH-I and Panbo-RPCH. A third octapeptide AKH (Locmi-AKH-III) was also found in

L. migratoria and a similar octapeptide (designated Phymo-AKH-III) occurs in pyrgomorphid and pamphagid grasshoppers, but an AKH-III is missing in S. gregaria. Subsequently, three research groups ultimately reported, in the same year, the presence of two octapeptides from the corpora cardiaca of the American cockroach, Periplaneta americana, that were structurally related to the locust AKHs and the crustacean RPCH. These two peptides were isolated on the basis of myotropic or heartbeat acceleration bioassays and are referred to as cardio acceleratory hormones (Peram-CAH-I and Peram-CAH-II), but they also produced hypertrehalosemia in the cockroach and represent the hypertrehalosemic hormones. These pioneering studies, along with numerous subsequent studies, demonstrated that there are, so far, about forty structurally related, but distinct, peptides with adipokinetic and hypertrehalosemic effects in the insects and one in crustacean (Table 3). The name adipokinetic hormone/red pigment-concentrating hormone (AKH/RPCH) family was coined for this general family of peptides, which likely encompasses the arthropods.

Chemistry of the AKH/RPCH Family

The members of the adipokinetic hormone/red pigment-concentrating hormone family share numerous structural features. They consist either of eight, nine or ten amino acids, depending on the insect species from which they are isolated. They are blocked by pyro-glutamate at the N-terminus and by an amide moiety at the C-terminus. Presumably, blocked termini prevent degradation of the neuropeptides by amino- and carboxypeptidase enzymes while circulating in the hemolymph. Aromatic amino acids, usually phenylalanine and tryptophan, always occupy positions 4 and 8, respectively, but aromatic amino acids can also occupy other positions. The peptides are usually neutral under physiological conditions, but a few have a negatively charged aspartate at position 7. Glycine is always present at position 9 as deduced from cDNA analysis of the precursor. The terminal glycine is converted to the amide moiety on the tryptophan in the octapeptides.

Some of the members of this peptide family have additional post-translational modifications besides the blocked termini. For example, two HrTH decapeptides are present in the corpora cardiaca of the stick insect, Carausius morosus; one of these decapeptides is glycosylated and has a unique C-glycosylation where the sugar is linked to the C-2 atom of the indole ring of tryptophan. Another unusual modification has been found in an AKH of the protea beetle, Trichostetha fascicularis: the corpora cardiaca contain two AKHs, one of which is an octapeptide with a phosphothreonine at position 6.

The relationships between individual AKH and HrTH peptides and insect species are complex. There are no clear rules concerning which peptide occurs in which order of insects. Several species within an order may share the same peptide and have other species-specific sequences, and the same peptide may be present in species of different orders. As described above, the two locust species,

S. gregaria and L. migratoria, share an identical decapeptide (Locmi-AKH-I); each species possesses a second, unique octapeptide (LocmiAKH-II; Schgr-AKH-II); and L. migratoria contains a third octapeptide (Locmi-AKH-III) that does not have a complement in S. gregaria. Cockroach species of the families Blattellidae and Blaberidae share a single hypertrehalosemic decapeptide hormone (Bladi-HrTH), whereas cockroaches of the family Blattidae contain two octapeptide hormones (Peram-CAH-I and -II). In addition, there is overlap between orders. Grybi-AKH is present in certain crickets and in species of Neuroptera, Dermaptera and Heteroptera. Peram-CAH-I and -II of the blattid cockroaches are also found in the Colorado potato beetle, Leptinotarsa decemlineata, and Peram-CAH-II is shared with the heteropteran bug, Pyrrhocoris apterus. Whereas Peram-CAH-I and -II mobilize glycogen from the fat body of the cockroach to increase hemolymph trehalose, the same or similar peptides increase hemolymph proline in beetles to serve as the major flight substrate.

Unlike the complex situation in insects, the crustaceans apparently possess only the single Panbo-RPCH peptide which has a chromatophorotropic effect. Panbo-RPCH has also been found in an insect species, the heteropteran stinkbug, Nezara viridula, where it has an adipokinetic effect.

Phylogenetic relationships of the HrTHs have been proposed for the cockroaches based on morphological, behavioral and physiological characters congruent with the distribution of the various structures of the HrTHs within the order.

Physiological Actions

The general physiological action of the adipokinetic and hypertrehalosemic hormones in insects is to elevate the hemolymph metabolites that are used by the muscles and other tissues as a source of energy, regardless of the nature of the metabolites. This is accomplished by stimulating the fat body, which is the hormone’s target tissue, to convert its stores of triacylglycerides or glycogen to diacylglycerides or trehalose, respectively, or to synthesize proline. The diacylglycerides, trehalose or proline are released from the fat body to increase their respective levels in the hemolymph. The same peptides that elevate diacylglycerides in locusts elevate trehalose when administered to cockroaches, and vice versa, and the hormones of locusts and cockroaches elevate proline in the Colorado potato beetle. The decision as to whether lipid-, carbohydrate-, or proline-mobilizing pathways are activated is a species-related function of the enzyme composition in the fat body.

Muscular activity for animal locomotion can involve either long-term or short-term events. Long-term activities might entail sustained, nonemergency actions such as migration or persistent searching for food, mates or shelter. Short-term activities might consist of local searching activities but may also require immediate, brief, emergency responses such as evading attack by a predator or defending a breeding territory. Longterm events require a steady supply of energy metabolites, whereas short-term events may be brief but intense, and, if successful, they can be followed by a period of recovery to replenish exhausted metabolites.

The adipokinetic hormones are oſten involved in prolonged, constant muscular activity such as migration. This is characteristically true for the locusts whose migratory behavior has been described since biblical times. Migration is a sustained flight activity that uses muscular oxidation of fatty acids to produce energy, since fatty acids deliver more energy per mole than carbohydrates. However, carbohydrate serves as the major source of muscular energy during initial flight, and lipid becomes the major source for energy as flight persists and becomes sustained. Based on differing physiological effects, it is speculated that the three AKHs may exert different regulatory actions on metabolite mobilization and use during the different stages of migration. Locmi-AKH-II is likely to be the major carbohydrate-mobilizing hormone that provides trehalose for initial flight; LocmiAKH-I is the major hormone responsible for fat mobilization during sustained flight and LocmiAKH-III may be responsible for regulating energy metabolism during rest. Furthermore, during lipid mobilization, AKH performs several distinct but related actions. In the fat body, AKH activates lipase for triacylglyceride degradation; this is achieved by binding of the AKH to a G-protein coupled receptor at the cell membrane, activation of adenylate cyclase resulting in the second messenger cAMP which, in conjunction with Ca2+, is responsible for lipase activation. In the hemolymph, AKH increases the lipid-carrying capacity of lipophorins (proteins) resulting in increased amounts of low-density lipophorin for shuttling lipids from the fat body to the muscles. At the flight muscle level, AKH increases the rate of lipid oxidation. Recent research on a number of terrestrial and aqueous heteropteran bugs that have various feeding patterns (plant sap sucking, predators, obligatory hematophagous), also established a lipid-based activity (flight and/or swimming) metabolism that is regulated by the respective AKHs of these insects.

By contrast, insects such as cockroaches, bees and flies use only carbohydrate (trehalose) as the primary source of energy for muscular activity and locomotion. These species do not migrate and lack the adipokinetic response, but they are faced with emergency situations of predator evasion, and in such cases, the hypertrehalosemic hormone mobilizes trehalose in response to the emergency. However, injections of hypertrehalosemic hormone show that significant elevation of the hemolymph trehalose may take as long as 10–30 min. This delay in elevating hemolymph carbohydrate is too long to significantly assist the insect in evading capture. Furthermore, the open circulatory system of insects does not efficiently direct circulating metabolites to the muscles in the manner of the closed circulatory system of vertebrate animals. Energy metabolites, such as trehalose, must constantly be maintained at high levels in the hemolymph to meet urgent, immediate demands. Hence, the role of the hypertrehalosemic hormone appears to be to replace depleted hemolymph trehalose and maintain it at high levels. Maintenance of high trehalose levels allows the insect to make quick responses to elude capture that may require only seconds, or at most, several minutes to conclude. If the insect is successful at escape, the hormone stimulates the degradation of fat body glycogen to restore the high trehalose levels by activating specifically the enzyme glycogen phosphorylase aſter the hormone has bound to a G-protein coupled receptor on the membrane of a fat cell and had activated a phospholipase C, resulting in the production of inositol trisphosphate and the release of Ca2+ from internal stores (influx of external Ca2+ is also activated by HrTH) which sets in motion a cascade of activation of kinases and, finally, glycogen phosphorylase. Removal of the hypertrehalosemic hormone does not affect the ability of such insects to be active for the short term (several minutes), but aſter exhaustion, lengthens their recovery time.

Tsetse flies and various beetle species fuel their flight metabolism by the partial oxidation of proline and the production of alanine. For continuous flight or replenishment of proline reserves in the fat body a unique system exists in these insects to synthesis proline: the respective AKHs activate a lipase in the fat body and the fatty acids that are liberated from triacylglyerols undergo β-oxidation, and the resulting acetyl CoA units are used in conjunction with alanine to synthesize proline. Alanine, which is derived from the partial oxidation of proline, is re-used for proline synthesis and can be viewed as a shuttle system for the transport of acetyl units.

Although the mobilization of energy for flight and other metabolically intense situations is likely the major function for the adipokinetic and hypertrehalosemic hormones, the hormones exhibit pleiotropic actions. The hypertrehalosemic hormones were isolated originally based on their cardioacceleratory action on the heart. This is a logical action for the hormone since elevated heartbeat rate would facilitate distribution of the energy metabolites throughout the body and assure their ready access to the muscles. In keeping with the stimulatory action of AKH on lipid degradation in locusts, lipid synthesis is inhibited by AKH. Other, less well characterized actions include: inhibition of RNA and protein synthesis related to vitellogenesis in locusts and crickets, and the stimulation in cockroaches of the oxidative capacity of mitochondria during fat body maturation, and of gene expression for a fat body cytochrome P450 related to lipid oxidation. These latter actions by the hormones may be equally important as their effects on mobilization of energy metabolites, but they are poorly elucidated because of insufficient research, and they cannot yet be placed into perspective as to their physiological significance. Other actions in which AKHs seem to be involved are an enhanced activation of the locust immune system and, possibly, in the activation of an antioxidant protection mechanism in potato beetles.

In summary, the adipokinetic-hypertrehalosemic-hyperprolinemic hormones constitute a family of peptides that are adapted to the individual biology of the insect species in which they are found. They display a unique relationship with their target tissue in that the hormone carries the endocrine message to the target tissue (fat body) to mobilize energy stores, but the target tissue determines which metabolic pathways are activated depending on the biology of the species. It is this biology that determines the nature of the muscular activity (prolonged—migration; brief—predator evasion) and its metabolic need for consuming carbohydrates, lipids or proline as a source of energy.

Table 3 Representative sequences of adipokinetic/hypertrehalosemic peptides from various insect orders

Order	Peptide name	Genus	Structure
			1	2	3	4	5	6	7	8	9	10	11
Odonata	Libau-AKH	Libellula, Pantala, Orthetrum	pGlu	Val	Asn	Phe	Thr	Pro	Ser	Trp	NH2	-	—
Odonata	Anaim-AKH	Anax, Aeshna	pGlu	Val	Asn	Phe	Ser	Pro	Ser	Trp	NH2	-	—
Odonata	Psein-AKH	Pseudagrion, Ischnura	pGlu	Val	Asn	Phe	Thr	Pro	Gly	Trp	NH2	-	—
Blattodea	Peram-CAH-Ia	Periplaneta, Blatta	pGlu	Val	Asn	Phe	Ser	Pro	Asn	Trp	NH2	-	—
Blattodea	Peram-CAH-IIa	Periplaneta, Blatta	pGlu	Leu	Thr	Phe	Thr	Pro	Asn	Trp	NH2	-	—
Blattodea	Bladi-HrTH	Blaberus, Nauphoeta	pGlu	Val	Asn	Phe	Ser	Pro	Gly	Trp	Gly	Thr	NH2
Mantodea	Emppe-AKH	Empusa, Sphodromantis	pGlu	Val	Asn	Phe	Thr	Pro	Asn	Trp	NH2	-	—
Phasmatodea	Carmo-HrTH	Carausius, Sipyloidea, Extatosoma	pGlu	Leu	Thr	Phe	Thr	Pro	Asn	Trp	Gly	Thr	NH2
Mantophasmatodea	Manto-AKH	Not known	pGlu	Val	Asn	Phe	Ser	Pro	Gly	Trp	NH2	-	—
Orthoptera, Caelifera	Locmi-AKH-I	Locusta, Schistocerca	pGlu	Leu	Asn	Phe	Thr	Pro	Asn	Trp	Gly	Thr	NH2
Orthoptera, Caelifera	Locmi-AKH-II	Locusta	pGlu	Leu	Asn	Phe	Ser	Ala	Gly	Trp	NH2	-	—
Orthoptera, Caelifera	Schgr-AKH-IIa	Schistocerca, Phymateus	pGlu	Leu	Asn	Phe	Ser	Thr	Gly	Trp	NH2	-	—
Orthoptera, Caelifera	Locmi-AKH-III	Locusta	pGlu	Leu	Asn	Phe	Thr	Pro	Trp	Trp	NH2	-	—
Orthoptera, Caelifera	Phymo-AKH-III	Phymateus	pGlu	Ile	Asn	Phe	Thr	Pro	Trp	Trp	NH2	-	—
Orthoptera, Ensifera	Grybi-AKHa	Gryllus, Acheta, Gryllodes	pGlu	Val	Asn	Phe	Ser	Thr	Gly	Trp	NH2	-	—
Orthoptera, Ensifera	=Schgr-AKH-II	Tettigonia, Decticus	pGlu	Leu	Asn	Phe	Ser	Thr	Gly	Trp	NH2	-	—
Isoptera	Micvi-CC	Microhodotermes	pGlu	Ile	Asn	Phe	Thr	Pro	Asn	Trp	NH2	-	—
Dermaptera	=Grybi-AKH	Labidura, Forficula	pGlu	Val	Asn	Phe	Ser	Thr	Gly	Trp	NH2	-	—
Hemiptera/Homoptera	Placa-HrTH	Platypleura, Munza, Cacama, Magicicada, Diceroprocta	pGlu	Val	Asn	Phe	Ser	Pro	Ser	Trp	Gly	Asn	NH2
Hemiptera, Heteroptera	Pyrap-AKH	Pyrrhocoris, Disdercus	pGlu	Leu	Asn	Phe	Thr	Pro	Asn	Trp	NH2	-	—
Hemiptera, Heteroptera	=Peram-CAH-II	Pyrrhocoris, Disdercus	pGlu	Leu	Thr	Phe	Thr	Pro	Asn	Trp	NH2	-	—
Hemiptera, Heteroptera	Corpu-AKH	Corixa	pGlu	Leu	Asn	Phe	Ser	Pro	Ser	Trp	NH2	-	—
Hemiptera, Heteroptera	Letin-AKH	Lethocerus	pGlu	Val	Asn	Phe	Ser	Pro	Tyr	Trp	NH2	-	—
Hemiptera, Heteroptera	Nepci-AKH	Nepa	pGlu	Leu	Asn	Phe	Ser	Ser	Gly	Trp	NH2	-	—
Neuroptera	=Grybi-AKH	Palpares	pGlu	Val	Asn	Phe	Ser	Thr	Gly	Trp	NH2	-	—
Coleoptera	Scade-CC-I	Scarabaeus, Gareta, Onitis	pGlu	Phe	Asn	Tyr	Ser	Pro	Asp	Trp	NH2	-	—
Coleoptera	Scade-CC-II	Scarabaeus, Gareta	pGlu	Phe	Asn	Tyr	Ser	Pro	Val	Trp	NH2	-	—
Coleoptera	Oniay-CC	Onitis	pGlu	Tyr	Asn	Phe	Ser	Thr	Gly	Trp	NH2	-	—
Coleoptera	=Peram-CAH-I	Leptinotarsa	pGlu	Val	Asn	Phe	Ser	Pro	Asn	Trp	NH2	-	—
Coleoptera	=Peram-CAH-II	Leptinotarsa	pGlu	Leu	Thr	Phe	Thr	Pro	Asn	Trp	NH2	-	—
Lepidoptera	Manse-AKHa	Manduca, Vanessa, Bombyx, Heliothisb	pGlu	Leu	Thr	Phe	Thr	Ser	Ser	Trp	Gly	NH2	—
Lepidoptera	Helze-HrTH	Heliothisb	pGlu	Leu	Thr	Phe	Ser	Ser	Gly	Trp	Gly	Asn	NH2
Hymenoptera	Tenar-HrTH	Tenthredo	pGlu	Leu	Asn	Phe	Ser	Thr	Gly	Trp	Gly	Gly	NH2
Hymenoptera	=Schgr-AKH-II	Xylocopa, Bombus	pGlu	Leu	Asn	Phe	Ser	Thr	Gly	Trp	NH2	-	—
Hymenoptera	=Grybi-AKH	Vespula, Vespa	pGlu	Val	Asn	Phe	Ser	Thr	Gly	Trp	NH2	-	—
Hymenoptera	=Manse-AKH	Apis	pGlu	Leu	Thr	Phe	Thr	Ser	Ser	Trp	Gly	NH2	—
Diptera	Phote-HrTH	Phormia, Drosophila	pGlu	Leu	Thr	Phe	Ser	Pro	Asp	Trp	NH2	-	—
Diptera	Anoga-AKH	Anopheles	pGlu	Leu	Thr	Phe	Thr	Pro	Ala	Trp	NH2	-	—
Diptera	Tabat-AKH	Tabanus	pGlu	Leu	Thr	Phe	Thr	Pro	Gly	Trp	NH2	-	—
Crustacea	Panbo-RPCH	Pandalus	pGlu	Leu	Asn	Phe	Ser	Pro	Gly	Trp	NH2	-	—

^a Note that the peptide in certain orders is identical. For example: Peram-CAH-I and -II of the Blattodea, Blattidae is also present in Coleoptera (Leptinotarsa); Schgr-AKH-II of Orthoptera, Caelifera is present in Orthoptera, Ensifera and in Hymenoptera (Xylocopa, Bombus); Grybi-AKH of Orthoptera, Ensifera is also present in Dermaptera, Neuroptera and Hymenoptera, etc.

^b Heliothis is revised to Helicoverpa.