Adipokinetic Hormones: Coupling between Biosynthesis and Release

291

Adipokinetic Hormones

Coupling between Biosynthesis and Release

ROB C. H. M. OUDEJANS,a LUCIEN F. HARTHOORN, JACQUES H. B. DIEDEREN, AND DICK J. VAN DER HORST

Biochemical Physiology Research Group, Utrecht University,NL-3584 CH Utrecht, the Netherlands

ABSTRACT: During long-distance flight of migratory locusts, the dramatic en-ergy demand of the flight muscles is controlled by three adipokinetic hormones(AKHs). These peptide hormones regulate the mobilization of lipid and carbo-hydrate stored in the fat body to serve as energy substrates for the flight mus-cles. Despite the relatively huge quantities of the three AKHs that are stored inthe corpora cardiaca, flight induces a differential 2–4-fold increase in themRNAs for the three hormones. Moreover, newly synthesized AKHs can be re-leased only during a restricted period of time, suggesting that by far most ofthe stored hormones are physiologically inactive. This raises the question ofhow the biosynthetic activity in the AKH-producing cells is coupled to theirsecretory activity. The present review discusses the potential mechanisms bywhich generation and release of mixtures of bioactive neurohormones are con-trolled and how peptidergic neuroendocrine cells cope with variations in phys-iological stimulation, with the AKH-producing cells serving as a model system.

THE ADIPOKINETIC HORMONES

Insect long-distance flight activity involves extremely high metabolic rates andinsect flight muscles are among the most energy-demanding tissues known. The en-ergy substrates needed for the flight muscles are stored in the fat body. Their mobi-lization is under control of adipokinetic hormones (AKHs). These peptide hormonesare synthesized and stored in the intrinsic neuroendocrine adipokinetic cells of theglandular lobes of the corpus cardiacum,1 also called adipokinetic cells.

To date, over 30 different AKHs are known from representatives of most insectorders.2–4 They are peptides consisting of 8 to 10 amino acids, N-terminally blockedby a pyroglutamate residue and C-terminally blocked by an amide group. All AKHscontain a tryptophan residue at position 8 and an aromatic amino acid residue at po-sition 4. The nonapeptide and decapeptide members have a glycine residue at posi-tion 9. Other characteristics of the various AKHs are less universal.3 We will focuson the AKHs of the migratory locust, Locusta migratoria, an internationally recog-nized model insect that is used in our research group for studying the regulation ofmetabolism during exercise.

Thirty years ago, the first physiological data indicating the presence of a lipidmobilizing hormone in the locusts L. migratoria and Schistocerca gregaria were ob-

aCorresponding author: phone, +31 30 2532898; fax, +31 30 2532837.e-mail: [email protected]

292 ANNALS NEW YORK ACADEMY OF SCIENCES

tained by Beenakkers5 and by Mayer and Candy,6 respectively. The primary struc-ture of this AKH (AKH-I, a decapeptide) was elucidated by Stone et al.7 andappeared to be identical for both locust species. Carlsen et al.8 and Gäde et al.9 ob-tained indications for the presence of another AKH in these locust species, which isdifferent for S. gregaria and L. migratoria. This AKH-II is an octapeptide differingin one amino acid residue between both locust species.10 In 1991, we have isolatedand sequenced yet another AKH from L. migratoria (AKH-III, an octapeptide).11

Until now, this locust is the only insect species in which three AKHs have beenfound. The primary structures of the three AKHs of L. migratoria are given in FIG-URE 1, using the acronym system as proposed by Raina and Gäde.12

All three AKHs are involved in the regulation of the mobilization of both lipidsand carbohydrates from the fat body, although their action is differential.13,14 Also,several other effects are known, such as inhibition of the synthesis of proteins, RNA,and fatty acids in the fat body.3 The physiological responses and the signal transduc-tion pathways of the AKHs in the target organ(s) of L. migratoria have beenreviewed recently.15,16

HOW CAN NEUROENDOCRINE CELLS COPE WITH VARIATION IN PHYSIOLOGICAL STIMULATION?

A supposed strategy for neuroendocrine cells to cope with variations in physio-logical stimulation implies the storage of hormones to provide a reservoir that canbe called upon to meet demands over a short time, without the necessity for abruptchanges in hormone biosynthesis.17–19 The closeness of the coupling of secretoryand biosynthetic activities in a particular neuroendocrine gland may depend to alarge degree on the relative magnitude of the amount of hormone that is present in astored form. A gland that has large stores of hormone can meet secretory demandsfor a longer period than a gland with a smaller store. An alternative to reliance on a(large) pool of stored hormone might be the immediate upregulation of the biosyn-thesis of hormone.18,20

Surprisingly little is known as to which alternative, or combination of alterna-tives, neuroendocrine cells adopt to cope with variations in physiological stimula-tion. To unravel this problem for peptidergic neuroendocrine cells, it is necessary tomeasure the stimulation delivered to the cells and the effects that this stimulation has(1) on the amount of hormonal neuropeptide released, (2) on the intracellular levelsof the specific mRNAs, the prohormones, and the neuropeptides, and (3) on the rateof synthesis of bioactive neuropeptides. Until now, no neuroendocrine systems areknown where all these parameters have been investigated in a comprehensive, quan-

FIGURE 1. Amino acid sequences of the adipokinetic hormones of the migratory lo-cust, Locusta migratoria, using the acronym system proposed by Raina and Gäde.12

293OUDEJANS et al.: ADIPOKINETIC HORMONES

titative way. In our opinion, the AKH-producing cells of the migratory locust offera good model system to study all these aspects.

The most important steps in the overall pathway for the biosynthesis and furthermetabolism of a peptide hormone are transcription, translation, processing, storage,release, and degradation. In FIGURE 2, this is schematically shown, based on ascheme by Morgan and Chubb.18 With respect to the adipokinetic cells, it is impor-tant to know which of the steps in this scheme are regulated in response to changesin physiological demand, with particular emphasis on the balances between biosyn-thesis, storage, and release of the AKHs.

BIOSYNTHESIS OF THE AKHS

All three AKHs are translated from separate mRNAs. The resulting preprohor-mones have a simple structure, consisting of a signal sequence, a monocopy AKHsequence, a dibasic processing site, and an AKH-associated peptide sequence, as de-duced from their cDNA sequences.21 After cotranslational cleavage of the signal se-quence, the resulting prohormones of AKH-I and -II dimerize at random by

FIGURE 2. A general scheme for the metabolism of a peptide hormone, based on ascheme of Morgan and Chubb.18


oxidation of their cysteine residues and give rise to two homodimeric (AKH-I/I andAKH-II/II) and one heterodimeric (AKH-I/II) precursors.11,22 This dimerization isa rather unique phenomenon that was first established for the AKHs of S. gregar-ia.23,24 Further proteolytic processing of the dimeric precursors in the secretorygranules of the AKH-producing cells of the corpus cardiacum results in the bioactivehormones and two homo- and one heterodimeric AKH precursor–related peptides(APRPs). The complete biosynthesis and processing of the AKHs, from amino acidsto bioactive hormones, takes less than 75 min.25 With respect to the biosynthesis ofAKH-III, experimental data on its processing are not yet available. As far as weknow, no dimerization of AKH-III prohormone occurs with those of AKH-I and -II.One may speculate that the two cysteine residues present in the AKH-III prohor-mone form an internal disulfide bond, giving rise to a cyclic prohormone and, afterprocessing, to a cyclic AKH-III-associated peptide.

In situ hybridization demonstrated that the sites for expression of the three AKHmRNAs are colocalized in the cell bodies of the AKH cells.21 Application ofimmuno-electron microscopical techniques showed that all three AKHs are co-localized in the secretory granules26 (also Harthoorn et al., in preparation). Northernblot analysis of mRNAs for AKH-preprohormones revealed that 1 h of flight activityresulted in an increase of the steady state levels of the mRNAs of all three AKH-preprohormones. This increase was differential since the levels for AKH-I and -IIwere about two times higher and for AKH-III even over four times higher than atrest.21 This observation is rather unexpected since only a very small fraction (1–2%)of the total store of AKHs is released during flight.27,28 The observed increase inmRNAs during flight may be related to the relatively small amounts of AKH avail-able for direct use, as discussed below, rendering upregulation vital. Besides, itshould be stressed that elevations in mRNA content may not always indicate a directincrease in protein synthesis.29

RELEASE OF THE AKHS

Flight is the only natural stimulus known to date for release of the AKHs.30 As aconsequence of their colocalization, the three AKHs will be released simultaneously,and also the corresponding APRPs will appear in the hemolymph by this exocytoticprocess. The resulting multifactorial signal consists of a complex mixture of pep-tides, likely with a multitude of effects on their targets.

The release of the AKHs by the adipokinetic cells appears to be subjected tomany regulatory (both stimulatory and inhibitory) substances.30 The various sub-stances can be of both neural and humoral origin. The neural regulation of the AKHrelease appears to be limited to about 15 secretomotor neurons in the protocerebrum,which send their axons via the nervi corporis cardiaci II into the glandular part of thecorpus cardiacum. Part of these neurons are locustatachykinin (Lom-TK)–immunopositive and part are FMRFamide-related peptide (FaRP)–immunopositive.Nonpeptidergic factors have not been established to be present in the neural fibersthat contact the AKH cells. Lom-TK-I and -II stimulate the AKH release in vitro31,32

and the FaRPs, SchistoFLRFamide and FMRFamide, inhibit the AKH release.33,34

Recently, we have found that locustamyoinhibiting peptide (Lom-MIP) has also aninhibitory effect on the release of AKHs (Harthoorn et al., in preparation). Humoral


regulation of the AKH release comprises components of both peptidergic nature(crustacean cardioactive peptide, CCAP35) and aminergic nature (octopamine,dopamine, serotonin36), which all stimulate the AKH release. The metabolic inter-mediate, the disaccharide trehalose, inhibits the release of AKH.37 Until now, therelative contribution of all these substances to the regulation of the AKH release invivo is not known and remains to be established. An extensive discussion of the reg-ulation of the AKH release is provided in a review by Vullings et al.30

PREFERENTIAL RELEASE OF NEWLY SYNTHESIZED AKHS

To study the release of AKHs, we used an in vitro system of corpora cardiaca sup-plied with a tritiated amino acid.38,39 Incorporation of radiolabel into the AKHs wasused to distinguish newly synthesized (radioactive) AKHs from older, preexisting(nonradioactive) AKHs. The ratio between the specific radioactivities of the releasedand the nonreleased AKHs was always >1.0, indicating that newly synthesized hor-mones are released preferentially (last-in, first-out principle). Since the percentageof newly synthesized AKHs that were released increased for a period of about 8 hand decreased thereafter, it is presumed that, after packaging of the prohormonesinto secretory granules and their processing to bioactive AKHs, some further matu-ration of the secretory granules is required before they can release their content. Thedecrease of released radioactive AKHs after 8 h suggests that secretory granules con-taining radioactive AKHs enter a nonreleasable pool of older secretory granules. Theexistence of a large nonreleasable pool of AKH-containing secretory granules stress-es the fact that determination of the total hormone content of a neuroendocrine struc-ture has only a limited physiological value; only the releasable amount of hormoneis relevant.

STORAGE OF THE AKHS

Studies on the total content of the three AKHs in the corpus cardiacum of larvalstages and adults of L. migratoria show a continuous increase of the amounts ofthree AKHs.40 In corpora cardiaca from adult animals usually used for experiments(12–16 days after their final molting), the molar ratio of AKH-I, -II, and -III is14:2:1.11,40 The ratio between AKH-I and -II remains constant throughout the lifecycle, whereas the amount of AKH-III is relatively lower during larval developmentthan in the adult stage.40 Interestingly, the ratio between AKH-I and -II in S. gregariais not constant.41 In the AKH cells of aging L. migratoria, not only the number ofsecretory granules increases, but also an increasing number of very large intracister-nal granules is found, representing stores of AKH prohormones.42,43

DEGRADATION OF THE AKHS

Although knowledge on the amounts of hormones that are released by neuro-endocrine cells after stimulation is useful to interpret experimental results, the bal-ance between hormone release and the rate of degradation of released hormones in


the hemolymph (or blood) will ultimately determine their effect on the target cells.Particularly when a mixture of released hormones will be degraded differentially, thechanging ratio between the hormones may dramatically alter their joint regulatoryeffects.

Chemical synthesis of radiolabeled AKHs with very high specific radioactivitiesallowed us to conduct studies on the degradation of the AKHs with physiologicaldoses of as low as 1.0 pmol per locust.44 All three AKHs are inactivated by endo-proteolytic cleavage of the N-terminal tripeptide pGlu-Leu-Asn; the resulting pep-tides have no adipokinetic activity. The degradation of the AKHs appeared to bedifferential; half-lives obtained for AKH-I, -II, and -III are 51, 40, and 5 min, respec-tively, in animals at rest, and 35, 37, and 3 min during flight. The initial ratio of thehormones in the corpus cardiacum will therefore change rather drastically upon re-lease, particularly during flight conditions. AKH-I seems to be the most importanthormone during prolonged flight (mobilization and utilization of lipids). AKH-IIpresumably is important only at the onset of flight (mobilization and utilization ofcarbohydrates), whereas AKH-III, due to its low content and very rapid degradation,possibly only acts in a modulatory way under nonflight conditions.16,44

FIGURE 3. Possible coupling between secretory activity and transcription activationof the AKH-producing cells in Locusta migratoria.


COUPLING BETWEEN BIOSYNTHESIS AND RELEASEOF THE AKHS

Since flight-induced release of AKHs coincides with upregulation of theirmRNAs,21 secretory activity of the AKH-producing cells appears to be coupled totranscription activation. The possible coupling between these two cellular processesmight exist at the level of signal transduction. When a peptide hormone (CCAP,Lom-TK) binds to its receptor on the AKH-producing cells, an intracellular cascadeof second messengers (for instance, cAMP32) can be activated to stimulate AKH re-lease by these cells. The same activated signal transduction route, or part of thatpathway, also can stimulate gene expression by acting through transcription factors.This is shown schematically in FIGURE 3.

Further research will focus on the question of whether the biosynthesis and re-lease of the AKHs are finely tuned processes.

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Adipokinetic Hormones: Coupling between Biosynthesis and Release