A new adipokinetic hormone (named Lom-AKH-111) was isolated from the glandular lobes of the corpora cardiaca of Locusta migratoria. At the N-terminus it is blocked by a 5-oxoproline (pyroglutamic acid) residue (< Glu). After enzymatic deblocking, the amino acid sequence of the N-terminus was partly established by automatic Edman degradation to be 1 < Glul-Leu-Asn-Phe-Thr-Pro-. Fast-atom-bombardment spectrometry (FAB-MS) revealed that the new hormone is an octapeptide, which is amidated at the C-terminus, and has a relative molecular mass of 1072. Based on the FAB-MS data the complete sequence is < Glu-Leu-Asn-Phe-ThrPro-Trp-Trp-NH,, which was confirmed by chemical synthesis. All characteristics from HPLC, FAB-MS and biological activity of the natural hormone and the synthetic peptide appeared to be identical. Although the structure of this new hormone resembles that of Lom-AKH-I ( < Glu-Leu-Asn-Phe-Thr-Pro-Asn-Trp-Gly-Thr-NH,), its amino acid sequence points to a completely different route for its biosynthesis, involving a third prohormone. High-[K+]-containing media can cause release of all three adipokinetic hormones in vitro. Interestingly, the new hormone is absent in another locust species, Schistocerca gregaria. Based on in vitro biosynthesis experiments the turnover for this hormone is very high, suggesting an important physiological function. Locusta migratoria is the first insect species in which three different adipokinetic hormones have been demonstrated.The first physiological evidence for the presence of compound(s) with adipokinetic activity in the corpora cardiaca (CC) of the locusts Locusta migratoria and Schistocerca gregaria was reported independently by Beenakkers [l] and by Mayer and Candy 121, respectively. From both species an identical adipokinetic hormone (AKH) was isolated (Lom-AKH-I; for nomenclature see [3]) and sequenced ( < Glu-LeuAsn-Phe-Thr-Pro-Asn-Trp-Gly-Thr-NH,) 141. Both species also contain a second adipokinetic hormone [5] and sequence analysis revealed that this hormone in Locusta (Lom-AKH-11) ( < Glu-Leu-Asn-Phe-Ser-Ala-Gly-Trp-NH,) differs in only one amino acid from that in Schistocerca (Scg-AKH-11) ( < Glu-Leu-Asn-Phe-Ser-Thr-Gly-Trp-NH2) [6].AKHs are synthesized and stored in the glandular lobe of the CC [7 -91 and are colocalized in the same secretory granCorrespondence to R.

Three distinct cDNAs encoding the preproadipokinetic hormones I, II, and III (prepro-AKH I, II, and III), respectively, of Locusta migratoria have been isolated and sequenced. The three L. migratoria AKH precursors have an overall architecture similar to that of other precursors of the AKH/red pigment-concentrating hormone (RPCH) family identified so far. The AKH I and II precursors of L. migratoria are highly homologous to the Schistocerca gregaria and Schistocerca nitans AKH precursors. Although the L. migratoria AKH III precursor appears to be the least homologous to the Manduca sexta, Drosophila melanogaster, and Carcinus maenas AKH/RPCH precursors, we favor the opinion that the L. migratoria AKH III precursor is evolutionary more related to the M. sexta, D. melanogaster, and C. maenas AKH/RPCH precursors than to the AKH I and II precursors of S. gregaria, S. nitans, or L. migratoria. In situ hybridization showed signals for the different AKH mRNAs to be co-localized in cell bodies of the glandular lobes of the corpora cardiaca. Northern blot analysis revealed the presence of single mRNA species encoding the AKH I precursor (ϳ570 bases), AKH II precursor (ϳ600 bases), and AKH III precursor (ϳ670 bases), respectively. Interestingly, flight activity increased steady-state levels of the AKH I and II mRNAs (ϳ2.0 times each) and the AKH III mRNA (ϳ4.2 times) in the corpora cardiaca.Three peptide hormones with hyperlipemic activity, the adipokinetic hormones I, II and III (AKH 1 I, II and III; see Table I) (1-3), are synthesized by the glandular neurosecretory cells of the corpora cardiaca (CC) of the migratory locust, Locusta migratoria. These peptides are members of a large family of structurally related but functionally diverse peptides (the AKH/RPCH family) (4). In the adult locust, the AKHs I and II are released into the hemolymph during flight and are involved in the mobilization of lipid and carbohydrate from the fat body to serve as energy substrates for the flight muscles (4 -7). Data on the release and functioning of AKH III are lacking so far. Isolation and characterization of CC peptides revealed that two other locust species, Schistocerca gregaria and Schistocerca nitans, each contain two AKHs that are mutually identical (1, 8), whereas Manduca sexta and Drosophila melanogaster each contain only one AKH (9, 10) (see Table I).Molecular biological studies have resulted in the characterization of the structure of the AKH/RPCH precursors (a signal peptide, AKH/RPCH, a Gly-(Lys/Arg)-Arg sequence, and an AKH/RPCH-associated peptide (AAP/RAP), in this order) of S. gregaria, S. nitans, M. sexta, D. melanogaster,.The biosynthesis of the AKHs in S. gregaria has been elucidated in detail by O'Shea and co-workers (13, 18 -21). The signal peptide is co-translationally removed from prepro-AKH, generating pro-AKH. Next, proteolytic processing, which is preceded by dimerization of two pro-AKHs (I/I, I/II, or II/II) via their single COOH-terminal Cys residues, gives rise to two AKHs (I and/or II) and one homo-or hete...

The adipokinetic cells are neuron-like unipolar cells, the cell bodies and cell processes of which are intermingled within the glandular part of the corpus cardiacum. In Schistocerca gregaria, they produce two adipokinetic hormones, AKH-I and -II, whereas in Locusta migratoria an additional hormone, AKH-III, is present. The three AKHs are produced by the same cells and are co-localized in secretory granules. The biosynthesis and processing of the AKH prohormones to the bioactive hormones, which has been elucidated in detail for AKH-I and -II in S. gregaria, takes less than 75 min and goes on continuously. In older locusts in particular, the adipokinetic cells contain intracisternal granules, widely dilated cisternae of the rough endoplasmic reticulum, which function as stores of prohormones of AKH-I and -II, not of AKH-III. The adipokinetic cells are subjected to regulation by a number of neural and humoral substances, neural influences coming from secretomotor cells in the lateral part of the protocerebrum. Flight activity is the only natural stimulus unequivocally shown to induce the release of AKHs, which in L. migratoria results in parallel secretion of all three AKHs. During secretory stimulation, young secretory granules containing newly synthesized hormones are preferentially released over older granules. Secretory stimulation is not accompanied by a clear increase in the levels of the AKH mRNAs and the AKH prohormones and in the rate of synthesis of the (pro-)AKHs. Apparently, a coupling between release and biosynthesis of the AKHs in the adipokinetic cells is very loose or does not even exist.

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Since concomitant release of structurally related peptide hormones with apparently similar functions seems to be a general concept in endocrinology, we have studied the dynamics of the lifetime of the three known adipokinetic hormones (AKHs) of the migratory locust, which control flight-directed mobilization of carbohydrate and lipid from fat body stores. Although the structure of the first member of the AKHs has been known for 20 years, until now, reliable data on their inactivation and removal from the hemolymph are lacking, because measurement requires AKHs with high specific radioactivity. Employing tritiated AKHs with high specific radioactivity, obtained by catalytic reduction with tritium gas of the dehydroLeu2 analogues of the AKHs synthesized by the solid-phase procedure, studies with physiological doses of as low as 1.0 pmol per locust could be conducted. The AKHs appear to be transported in the hemolymph in their free forms and not associated with a carrier protein, despite their strong hydrophobicity. Application of AKHs in their free form in in vivo and in vitro studies therefore now has been justified. We have studied the degradation of the three AKHs during rest and flight. The first cleavage step by an endopeptidase is crucial, since the resulting degradation -products lack any adipokinetic activity. Half-lives for AKH-I, -II and -m were 51, 40, and 5 min, respectively, for rest conditions and 35, 37, and 3 min, respectively, during flight.

Immunoreactivity of granules containing secretory material in the adipokinetic cells of the insect Locusta migratoria was studied using antisera specific for the adipokinetic hormone-associated peptides (AAP) I, II and III. Immunocytochemical detection of these associated peptides represents a new strategy for studying the intracellular location of the adipokinetic hormones and their prohormones. Fixation with 2% glutaraldehyde and 2% formaldehyde with low-temperature embedding in Lowicryl HM20 allowed highly selective immunogold labelling of both secretory and intracisternal granules. All three associated peptides were co-localized in secretory granules. This indicates that also all three adipokinetic hormones can be co-localized in these granules, which was confirmed by experiments in which, after secretory stimulation, adipokinetic hormone III was released from the adipokinetic cells together with adipokinetic hormones I and II. The immunopositivity of the intracisternal granules was similar to that of the secretory granules, although with the exception that the intracisternal granules did not show any specific reaction with anti-AAP III. The presence of AAP I and AAP II in intracisternal granules indicates that these granules only function as stores of adipokinetic prohormones I and II and not of adipokinetic prohormone III. The observed differences in storage in intracisternal granules among the three adipokinetic prohormones suggest differences in physiological significance of the three adipokinetic hormones in L. migratoria.

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