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Heterodimeric fly glycoprotein hormone-alpha2 (GPA2) and glycoprotein hormone-beta5 (GPB5) activate fly leucine-rich repeat-containing G protein-coupled receptor-1 (DLGR1) and stimulation of human thyrotropin receptors by chimeric fly GPA2 and human GPB5.

Glycoprotein hormones play important roles in thyroid and gonadal function in vertebrates. The glycoprotein hormone alpha-subunit forms heterodimers with different beta-subunits to activate TSH or gonadotropin (LH and FSH) receptors. Recent genomic analyses allowed the identification of another alpha-subunit, GPA2, and another beta-subunit, GPB5, in human, capable of forming heterodimers to activate TSH receptors. Based on comparative genomic searches, we isolated the fly orthologs for human GPA2 and GPB5, each consisting of 10 cysteine residues likely involved in cystine-knot formation. RT-PCR analyses in Drosophila melanogaster demonstrated the expression of GPA2 and GPB5 at different developmental stages. Immunoblot analyses further showed that fly GPA2 and GPB5 subunit proteins are of approximately 16 kDa, and coexpression of these subunits yielded heterodimers. Purified recombinant fly GPA2/GPB5 heterodimers were found to be glycoproteins with N-linked glycosylated alpha-subunits and nonglycosylated beta-subunits, capable of stimulating cAMP production mediated by fly orphan receptor DLGR1 but not DLGR2. Although the fly GPA2/GPB5 heterodimers did not activate human TSH or gonadotropin receptors, chimeric fly GPA2/human GPB5 heterodimers stimulated human TSH receptors. These findings indicated that fly GPA2/GPB5 is a ligand for DLGR1, thus showing the ancient origin of this glycoprotein hormone-seven transmembrane receptor-G protein signaling system. The fly GPA2 also could form heterodimers with human GPB5 to activate human TSH receptors, indicating the evolutionary conservation of these genes and suggesting that the GPA2 subunit may serve as a scaffold for the beta-subunit to activate downstream G protein-mediated signaling.

Amino Acid Sequence↗

[Molecular mechanism of diapause in Bombyx mori. 2. Diapause determination and expression of diapause hormone gene in pupal stage].

Embryonic diapause in Bombyx is induced by diapause hormone (DH), which is produced in the suboesophageal ganglion from a precursor protein. We have cloned the DH cDNA into pBluescript KS vector that containing T7 RNA promoter can be used to synthesize nonspecific RNA. DH cRNA was synthesized and used as internal control. We quantitatively measured DH mRNA at SG of day 3 pupae by Northern hybridization analysis and suggested that the embryonic diapause of Bombyx in next generation was determined by DH mRNA content in SG.

Animals↗

The fork head product directly specifies the tissue-specific hormone responsiveness of the Drosophila Sgs-4 gene.

Here we describe the identification of four binding sites of secretion enhancer binding protein 2 (SEBP2) in the regulatory region of the Drosophila salivary gland secretion protein gene 4 (Sgs-4) and show that despite these sites' correspondence with previously described Broad-Complex protein binding sites, SEBP2 is a Broad-Complex-independent factor encoded by the region-specific homeotic gene fork head (fkh). Two of the Fork head/SEBP2 binding sites are located within an ecdysone response unit which controls the tissue- and stage-specific responses of Sgs-4 to the steroid hormone 20-hydroxyecdysone. We demonstrate that these binding sites are relevant to the transcriptional activation of Sgs-4 and show that Fork head also binds to the Sgs-4 ecdysone response unit in vivo. Aside from being involved in the control of decisions during embryonic development, fkh thus participates directly in the control of specialized functions of differentiated cells at later stages of development.

Animals↗

Alternative farnesoid structures induce different conformational outcomes upon the Drosophila ortholog of the retinoid X receptor, ultraspiracle.

In view of recent studies that the ligand-binding pocket of the Drosophila melanogaster nuclear hormone receptor, ultraspiracle (dUSP), is a necessary component of dUSP-dependent transcriptional activation by methyl epoxyfarnesoate, we have assessed qualitative differences in the effect of farnesoid and dodecanoid compounds on receptor conformation and transcriptional activation. Farnesoids possessing terminal alcohol, aldehyde, acid, ester and/or epoxide moieties induced different changes in the local environment of the ligand-binding pocket, as monitored by the change each induced in the fluorescence of the two tryptophan residues existing in dUSP (that are situated 10 residues apart on the alpha-helix 5 that forms one lining of ligand-binding pocket). Similarly, each compound differed in the extent that it promoted an increase in anisotropy (dimerization state) of the receptor. Dodecanoid derivatives were much weaker in causing such effects. Methyl expoxyfarnesoate (insect juvenile hormone III) exhibited the greatest biological activity to increase transcription of a DR12JHECore reporter construct in transfected Sf9 cells, even though it did not exert the most suppression of USP fluorescence nor exert the greatest increase in USP anisotropy. In a comparison of farnesoid derivatives possessing the three side branches either as all methyl groups (JH III), or one of the side branches as ethyl (JH II), or two of the side branches as ethyl (JH I), the JH III and JH I were more similar to each other in the fluorescence suppression and in vivo morphogenetic activity than either was to JH II, evidencing that dUSP does not sense JH II as a structural 'intermediate' between JH III and JH I. Ligand-binding domains of vertebrate retinoid X receptors respond to agonists by repositioning alpha-helix 12 to the edge of a hydrophobic groove, and there with the groove jointly forms a coactivator binding surface. When alpha-helix 12 in dUSP was mutated to place two signaling tryptophan residues its C-terminus, fluorescence signaling indicated that upon dUSP binding of methyl epoxyfarnesoate, the alpha-helix 12 was repositioned differently than what occurred upon binding of non-JH farnesoids. These leads on alternative ligand-induced conformations that dUSP can adopt provide a foundation for commercial development of synthetic molecules that induce specific dUSP conformations, and for identification of in vivo conditions under which endogenous molecules may exert these conformational outcomes to this receptor.

Animals↗

Regulation of prothoracicotropic hormone release by juvenile hormone in the penultimate and last instar larvae of Mamestra brassicae.

The penultimate instar larvae of Mamestra brassicae have one ecdysteroid peak 2 days after ecdysis which induces larval ecdysis. Allatectomy deleted this peak and caused a larger peak 7 days later just before precocious pupation, in a pattern similar to the ecdysteroid titer of the last instar. Application of juvenile hormone (JH) immediately after allatectomy restored the normal larval pattern and larval ecdysis. The brain of the normal Day 1 or 2 penultimate instar larva had higher prothoracicotropic hormone (PTTH) activity than that of the allatectomized larva. Therefore, JH in the penultimate instar activates the brain to secrete and/or produce PTTH. Allatectomy of last instar larvae prolonged the period from gut purge to pupation by causing a delay of the ecdysteroid peak necessary for pupation. Application of JH restored the normal timing of ecdysteroid release. Thus, JH is important for the occurrence of the ecdysteroid surge on Day 7, since its absence delayed this surge by a day.

Animals↗

Inputs to the hormonal control of egg development in Rhodnius prolixus.

This paper re-examines existing data on the environmental inputs governing egg production in Rhodnius prolixus. Feeding has a direct effect on egg production such that the product of the unfed weight of the female times the weight of the blood meal is a good predictor of the number of eggs produced. Mating modifies this input, so that mated females produce more eggs. Egg production is governed by the corpus allatum, and indirect evidence suggests that the number of eggs producted by a female is a function of the length of time that juvenile hormone is secreted by the corpus allatum. The input which determines the times at which the corpus allatum is switched off originates in the stretch induced by the amount of the meal remaining in the crop, modified by the matedness status of the female. The precise nature of the sensors detecting stretch is not yet clear, but the integrity of the dorsal aorta is essential to the transmission of the information. These data are related to the survival strategy for Rhodnius.

Animals↗

[Adipocytokins, obesity and development of type 2 diabetes].

Normal metabolic balance is maintained by a complex homeostatic system involving multiple tissues and organs. Acquired or inherited defects associated to environmental factors in any part of this system can lead to metabolic disorders such as the syndrome X which is presently a frequent syndrome in industrialized countries. It is characterized by a cluster of risk factors of atherosclerosis including insulin resistance, hyperinsulinemia, impaired glucose tolerance or type 2 diabetes, hypertension, dyslipidemia, and coagulation abnormalities. Its pathophysiology is likely to involve insulin resistance at the level of both skeletal muscle and visceral adipose tissue and altered fluxes of metabolic substrates between these tissues that in turn impair liver metabolism. Therapeutic intervention favours at present diet and exercise prescriptions. In addition, if necessary, specific treatment of the metabolic disorders is required. In the treatment of insulin resistance, new promising drugs are likely to be used in the next future. In this regard, adipose tissue, once thought to function primarily as a passive depot for the storage of excess lipid, is now understood to play a much more active role in metabolic regulation, secreting a variety of metabolic hormones and actively functioning to prevent deleterious lipid accumulation in other tissues and to modulate the insulin resistance. Here, we review new advances in our understanding of mechanisms leading to insulin resistance and type 2 diabetes from the perspective of the role and interactions of recently identified adipocyte-specific chemical messengers, the adipocytokines, such as adiponectin, tumor necrosis factor-alpha, interleukin 6, and resistin.

Adiponectin↗

A reappraisal of the hormonal regulation of larval fat body histolysis in female Drosophila melanogaster.

The histolysis of larval fat body cells in adult female Drosophila melanogaster was examined in wild type and mutant animals. The fat body cells of wild type (Canton-S), apterous56f homozygotes, apterous78jts homozygotes and heterozygotes, apterous4/+, ecdysoneless1 homozygotes and heterozygotes all underwent histolysis normally during the 72 h following adult eclosion. Only in the case of ap4/ap4 adults did the cells fail to histolyze normally. The fat body cells of both diapausing and non-diapausing wild type females underwent histolysis at the same rate. Attempts to demonstrate histolysis in vitro were unsuccessful, even in the presence of juvenile hormones (JHs), larval ring glands, or adult ovaries. In all strains other than the ap4 homozygotes, a significant proportion of larval fat body cells were dead at any time while the ap4/ap4 animals, almost all cells remained viable. It is postulated that fat body cell lysis following eclosion is not a JH-mediated event, but is elicited by an as yet unidentified factor(s), possibly originating in the ovary.

Adipose Tissue↗

Adipokinetic hormones. Coupling between biosynthesis and release.

During long-distance flight of migratory locusts, the dramatic energy demand of the flight muscles is controlled by three adipokinetic hormones (AKHs). These peptide hormones regulate the mobilization of lipid and carbohydrate stored in the fat body to serve as energy substrates for the flight muscles. Despite the relatively huge quantities of the three AKHs that are stored in the corpora cardiaca, flight induces a differential 2-4-fold increase in the mRNAs for the three hormones. Moreover, newly synthesized AKHs can be released only during a restricted period of time, suggesting that by far most of the stored hormones are physiologically inactive. This raises the question of how the biosynthetic activity in the AKH-producing cells is coupled to their secretory activity. The present review discusses the potential mechanisms by which generation and release of mixtures of bioactive neurohormones are controlled and how peptidergic neuroendocrine cells cope with variations in physiological stimulation, with the AKH-producing cells serving as a model system.

Amino Acid Sequence↗

Helicostatins: brain-gut peptides of the moth, Helicoverpa armigera (Lepidoptera: Noctuidae).

Gene expression and immunolocalisation studies have determined that the helicostatins are brain-gut peptides in larvae of the lepidopteran, Helicoverpa armigera. Mapping of the distribution of these peptides in the nervous system and alimentary canal has provided evidence for multifunctional regulatory roles. In situ hybridisation studies have shown that the helicostatin precursor gene is expressed in neurones of the central and stomatogastric nervous systems, and endocrine cells of the midgut demonstrating that the helicostatins are true brain-gut peptides. Antisera raised against Leu-callatostatin 3 (ANRYGFGL-NH(2)), a peptide isolated from the blowfly, Calliphora vomitoria was used to map the distribution of allatostatin-like immunoreactive (Ast-ir) material in H. armigera to elucidate possible functions of the helicostatins. In situ hybridisation studies verified that the helicostatin precursor gene is expressed in neurones shown to contain Ast-ir, providing strong evidence that the Ast-ir material is helicostatins. Extensive immunoreactive axonal projections into complex regions of neuropile indicate that the helicostatins may have a neuromodulatory role in the brain and segmental ganglia of the ventral nerve cord. The presence of large amounts of immunoreactive material in axons within the corpora cardiaca (CC) and transverse nerves of the perisympathetic nervous system, two known neurohaemal organs, provides evidence for a neurohormonal role. The corpora allata (CA) were innervated only sparsely by Ast-ir axons suggesting that the CA are not a neurohaemal release site or a target. Thus, it is unlikely that the helicostatins regulate juvenile hormone (JH) biosynthesis or release. Ast-ir axons extended from the frontal ganglion through the recurrent nerve and many branches were closely associated with muscles of the foregut, stomodeal valve, and anterior midgut, implicating helicostatins in regulation of foregut motility. Ast-ir material was also present in nerves associated with muscles of the pyloric valve and rectum, and in endocrine cells of the midgut.

Animals↗

Characterisation of neuropeptides of the AKH/RPCH-family from corpora cardiaca of Coleoptera.

Extracts of corpora cardiaca from two members of the family Tenebrionidae. Zophobas rugipes and Tenebrio molitor, from one member of the Chrysomelidae, Leptinotarsa decemlineata, and from three members of the Scarabaeidae, Pachnoda marginata, P. sinuata and Melolontha hippocastani, were assayed for adipokinetic and hypertrehalosaemic activity in acceptor locusts (Locusta migratoria) and cockroaches (Periplaneta americana), respectively. All corpus cardiacum material tested, except that from the cockchafer, M. hippocastani, gave positive bioassay results. Biological activities of corpus cardiacum extracts from all species investigated can be resolved on reversed-phase high performance liquid chromatography (RP-HPLC). Gland extracts from the two tenebrionid species each show a single peak of biological activity associated with a single peak of UV absorbance having an identical retention time in both species. The two biologically active fractions from the corpora cardiaca of the potato beetle, L. decemlineata, coelute with exogenous (synthetic) hypertrehalosaemic hormones I and II of the American cockroach. The two species of the genus Pachnoda contain two active compounds in their glands; compound I of each species is more abundant and elutes just ahead of the (synthetic) hypertrehalosaemic hormone of the cockroach Blaberus discoidalis. The gland material of M. hippocastani exhibits an absorbance peak with the same retention time as the major peak from the Pachnoda-species; however, this peak material does not elicit biological activity in the assays used here. After fractionation by RP-HPLC the main biologically active compounds were subjected to amino acid analyses. All factors are peptidic and contain 8 amino acid residues.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗

N-terminal modifications to AKH-I from Locusta migratoria: assessment of biological potencies in vivo and in vitro.

To investigate the receptor tolerances to N-terminal variation, novel analogues to Locusta AKH-I (adipokinetic hormone) have been synthesized with modifications at the N-terminus. Analogues were made where the N-terminal pyroglutamyl residue was spaced further from the remainder of the molecule by the insertion of glycine residues between either pGlu1 and Leu2 (Gly1a-AKH-I, or Leu2 and Asn3 (Gly2a-AKH-I and Gly2ab-AKH-I). Other modified hormones with N-terminal extensions were: (Ahx)n-AKH-I (Ahx. aminohexanoic acid); HPP(Ahx)n-AKH-I (HPP. hydroxyphenyl propionate) and Ac(Ahx)n-AKH-I (where n = 0-3). Finally, acetylated and non-acetylated amino acids were substituted for pGlu1: Glu, Pro, Ala and Tyr. The effects of these modifications on biological potency were tested in the lipid mobilization assay in vivo and acetate uptake assay in vitro. The potency of AKH-I was reduced much more by insertion of glycine between pGlu1 and Leu2, than between Leu2 and Asn3, perhaps suggesting that a hydrophobic residue is required adjacent to the pGlu for biological activity. In addition, a residue N-terminal to Leu2 is necessary for activity (i.e., [despGlu]-AKH-I is inactive) unless the free N-terminus is acetylated: Ac[despGlu]-AKH-I is active, but has low potency. The potencies of HPP(Ahx)0-3-AKH-I, Ac(Ahx)1-3-AKH-I and glycine-inserted analogues decreased consistently with increasing extension of the N-terminus away from the remainder of the molecule. However, potencies of the unblocked (Ahx)n-AKH-I analogues did not, and potency in either assay did not appear related to the number of aminohexanoic residues. Similarly, while hormonal activity was retained by substitution of pGlu1 by Tyr, Pro, Ala or Glu in both assays, acetylation of the resulting analogues did not provide a consistent increase in potency, but actually decreased for AcGlu1-AKH-I compared with its unblocked analogue. HPP1-AKH-I was the most potent of the modified peptides tested, with almost the same potency in the assay in vitro as the natural peptide.

Acetates↗

Juvenile hormone inhibition of gene expression for cytochrome P4504C1 in adult females of the cockroach, Blaberus discoidalis.

The regulation and pattern of gene expression for cytochrome P4504C1 was measured in the fat body of adult females of the cockroach, Blaberus discoidalis. The level of CYP4C1-mRNA was high at adult emergence but disappeared after 4 days of adult life. In starved females, CYP4C1-mRNA levels declined by day 4 but increased steadily thereafter; by 25 days, the levels were nearly twice those observed at eclosion. Both the rapid early disappearance of the transcript and the starvation-related increase failed to occur following decapitation. Allatectomy also prevented the disappearance of CYP4C1-mRNA at day 4, and treatment of decapitated females with methoprene (JHA) stimulated a 70% decrease in transcript within 24 h. Injection of synthetic Blaberus hypertrehalosemic hormone (HTH) increased CYP4C1-mRNA by six-fold in the fat body of both intact and decapitated females. CYP4C1-mRNA in the fat body of males did not respond to JHA treatment. The dynamics of CYP4C1-mRNA in the fat body of females could be explained based on an inhibition of CYP4C1 expression by JH that was overcome by HTH.

Age Factors↗

Uptake of horseradish peroxydase by the testis of locusta migratoria during the last larval instar; relation with variations of ecdysteroid levels in haemolymph.

By using horseradish peroxydase (HRP) as a tracer, it is shown that the gonial region of the locust testis is an "open" compartment which is almost always freely penetrated by the tracer. During the last larval instar, however, the penetration of HRP decreases and ceases at the time when high levels of ecdysteroids are detected in the haemolymph by radioimmunoassay. A cause and effect relationship between tracer uptake and hormonal level could not be demonstrated by the experiments carried out up to now. From ultrastructural observations of the testis, it is concluded that the temporary isolation of the gonial compartment is not based upon any morphological structure which could act as a barrier. Penetration of the macromolecule is considered as the expression of an active uptake by the testis and the short period of nonpenetrability as a state of inertia whose significance remains to be discovered.

Animals↗

Effects of social environment and worker mandibular glands on endocrine-mediated behavioral development in honey bees.

Previous studies suggest that older honey bee workers possess an inhibitory signal that regulates behavioral development in younger bees. To study how this inhibitor is transmitted, bees were reared for 7 days in double-screen cages, single-screen cages, or unrestricted in a typical colony (control bees). Double-screen cages prevented physical contact with colony members while single-screen cages allowed only antennation and food exchange. Bees reared in double-screen cages showed accelerated endocrine and behavioral development; they had significantly higher rates of juvenile hormone biosynthesis and juvenile hormone titers than did control bees and also were more likely to become precocious foragers. Relative to the other two groups, bees reared in single-screen cages showed intermediate juvenile hormone biosynthesis rates and titers, and intermediate rates of behavioral development. These results indicate that physical contact is required for total inhibition. We also began to test the hypothesis that worker mandibular glands are the sources of an inhibitory signal. Old bees with mandibular glands removed were significantly less inhibitory towards young bees than were sham-operated and unoperated bees. These results suggest that an inhibitor is produced by the worker mandibular glands.

Animals↗

Multiple phosphorylation of ribosomal protein S6 and specific protein synthesis are required for prothoracicotropic hormone-stimulated ecdysteroid biosynthesis in the prothoracic glands of Manduca sexta.

Prothoracicotropic hormone (PTTH)-stimulated protein phosphorylation leads to ecdysteroidogenesis (molting hormone biosynthesis) in the prothoracic glands of the tobacco hornworm, Manduca sexta. The phosphorylation of 34 and 50 kDa peptides (p34 and p50) paralleled the increase in ecdysteroidogenesis, and the dephosphorylation of p34 and p50 preceded a decrease in ecdysteroidogenesis. Inhibition by rapamycin of p34, but not p50, phosphorylation prevented PTTH-stimulated ecdysteroidogenesis in a dose-dependent manner, suggesting that p34 phosphorylation is requisite for PTTH-stimulated ecdysteroidogenesis. Two proteins whose synthesis was rapidly stimulated by PTTH were p50 and p70. The time-course of PTTH-stimulated synthesis of p50 paralleled that of p34 phosphorylation and that of ecdysteroidogenesis. Rapamycin inhibited PTTH-stimulated synthesis of p50 and p70, suggesting that specific protein synthesis is also required for PTTH-stimulated ecdysteroidogenesis, confirming the results of Rybczynski and Gilbert [(1994) Insect Biochem. Molec. Biol. 24, 175-189], and that p34 phosphorylation may regulate the downstream synthesis of p50 and p70, possible key regulatory proteins leading to ecdysteroidogenesis. Results from two-dimensional (2D)-PAGE analysis of the ribosomal proteins purified from prothoracic glands, demonstrated that p34 is indeed ribosomal S6, and is phosphorylated at up to five sites (P1-5) upon PTTH stimulation. The multiple phosphorylation of S6 was inhibited completely by rapamycin as shown in 2D gel maps, further confirming that p34 is ribosomal protein S6. Temporal analysis of PTTH-stimulated S6 phosphorylation by 2D-PAGE revealed that phosphorylation of S6 at the P1 site was temporally correlated with the initiation of ecdysteroidogenesis, and that multiple phosphorylation at all five sites (P1-5) was correlated with the maximal synthesis of ecdysteroids. Dephosphorylation of S6 was accompanied by a decrease in ecdysteroidogenesis. These data demonstrate that p34 is ribosomal protein S6 and that both the phosphorylation of S6 and specific protein synthesis are required for PTTH-stimulated ecdysteroidogenesis in the prothoracic gland.

Animals↗

Lipid transport biochemistry and its role in energy production.

Recent advances on the biochemistry of flight-related lipid mobilization, transport, and metabolism are reviewed. The synthesis and release of adipokinetic hormones and their function in activation of fat body triacylglycerol lipase to produce diacylglycerol is discussed. The dynamics of reversible lipoprotein conversions and the structural properties and role of the exchangeable apolipoprotein, apolipophorin III, in this process is presented. The nature and structure of hemolymph lipid transfer particle and the potential role of a recently discovered lipoprotein receptor of the low-density lipoprotein receptor family, in lipophorin metabolism and lipid transport is reviewed.

Animals↗

Peptide-immunocytochemistry of neurosecretory cells in the brain and retrocerebral complex of the sphinx moth Manduca sexta.

Antisera against a variety of vertebrate and invertebrate neuropeptides were used to map cerebral neurosecretory cells in the sphinx moth Manduca sexta. Intense immunoreactive staining of distinct populations of neurosecretory cells was obtained with antisera against locust adipokinetic hormone, bovine pancreatic polypeptide, FMRFamide, molluscan small cardioactive peptide (SCPB), leucine-enkephalin, gastrin/cholecystokinin, and crustacean beta-pigment dispersing hormone (beta PDH). Other antisera revealed moderate to weak staining. Each type of neurosecretory cell is immunoreactive with at least one of the antisera tested, and most of these neurons can be identified anatomically. The staining patterns provide additional information on the organization of cerebral neurosecretory cells in M. sexta. Based upon anatomical and immunocytochemical characteristics, 11 types of neurosecretory cells have been recognized in the brain, one type in the suboesophageal ganglion, and one in the corpus cardiacum. Extensive colocalization experiments show that many neurosecretory cells are immunoreactive with several different antisera. This raises the possibility that these cells may release mixtures of neuropeptides into the hemolymph, as has been demonstrated in certain other systems. The immunocytochemical data should be helpful in efforts to identify additional peptide neurohormones released from the brain of this and other insects.

Animals↗