Local Juvenile Hormone activity regulates gut homeostasis and tumor growth in adult Drosophila.

Hormones play essential roles during development and maintaining homeostasis in adult organisms, regulating a plethora of biological processes. Generally, hormones are secreted by glands and perform a systemic action. Here we show that Juvenile Hormones (JHs), insect sesquiterpenoids synthesized by the corpora allata, are also synthesized by the adult Drosophila gut. This local, gut specific JH activity, is synthesized by and acts on the intestinal stem cell and enteroblast populations, regulating their survival and cellular growth through the JH receptors Gce/Met and the coactivator Tai. Furthermore, we show that this local JH activity is important for damage response and is necessary for intestinal tumor growth driven by activating mutations in Wnt and EGFR/Ras pathways. Together, our results identify JHs as key hormonal regulators of gut homeostasis and open the possibility that analogous hormones may play a similar role in maintaining vertebrate adult intestinal stem cell population and sustaining tumor growth.

Quantity does matter. Juvenile hormone and the onset of vitellogenesis in the German cockroach.

We aimed to elucidate why cockroaches do not produce vitellogenin in immature stages, by studying the appearance of vitellogenin mRNA in larvae of Blattella germanica. Treatment of female larvae in any of the last three instars with 1 microg of juvenile hormone (JH) III induces vitellogenin gene transcription, which indicates that the fat body is competent to transcribe vitellogenin at least from the antepenultimate instar larvae. In untreated females, vitellogenin production starts on day 1 after the imaginal molt, when corpora allata begin to synthesize JH III at rates doubling the maximal of larval stages. This coincidence suggests that the female reaches the threshold of JH production necessary to induce vitellogenin synthesis on day 1 of adult life. These data lead to postulate that larvae do not synthesize vitellogenin simply because they do not produce enough JH, not because their fat body is incompetent.

Screening of antifeedant activity in brain extracts led to the identification of sulfakinin as a satiety promoter in the German cockroach. Are arthropod sulfakinins homologous to vertebrate gastrins-cholecystokinins?

The feeding cycle of the adult female cockroach Blattella germanica parallels vitellogenesis. The study of the mechanisms that regulate this cycle led us to look for food-intake inhibitors in brain extracts. The antifeedant activity of brain extracts was tested in vivo by injecting the extract and measuring the carotenoids contained in the gut from carrot ingested after the treatment. By HPLC fractionation and tracking the biological activity with the carrot test, we isolated the sulfakinin EQFDDY(SO3H) GHMRFamide (Pea-SK). A synthetic version of the peptide inhibited food intake when injected at doses of 1 microg (50% inhibition) and 10 microg (60% inhibition). The sulfate group was required for food-intake inhibition. These biological and structural features are similar to those of the gastrin-cholecystokinin (gastrin-CCK) family of vertebrate peptides. However, heterologous feeding assays (human CCK-8 tested on B. germanica, and Pea-SK tested on the goldfish Carassius auratus) were negative. In spite of this, alignment and cluster analysis of these and other structurally similar peptide families suggest that sulfakinins and gastrin-CCKs are homologous, and that mechanisms of feeding regulation involving these regulatory peptides may have been conserved during evolution between insects and vertebrates.

Modulation of cardiac rhythm by allatostatins in the cockroach Blattella germanica (L.) (Dictyoptera, Blattellidae).

Cardiac rhythm was measured in Blattella germanica females during the reproductive cycle. The rate increased from day 0 to 1, remained constant during the vitellogenic period and fell by about 20% during the period of oothecal transport. The effects of allatostatins, allatostatin analogues and corazonin were tested on semi-isolated heart preparations. Allatostatins showed a rapid, reversible and dose-dependent cardioinhibitory activity. Blattella allatostatin 1 (BLAST-1: LYDFGL-NH(2)), was the most active, eliciting 76% inhibition at 10(-7) M and even 19% inhibition at 10(-9) M. BLAST-2 (DRLYSFGL-NH(2)), BLAST-3 (AGSDGRLYSFGL-NH(2)) and BLAST-4 (APSSAQRLYGFGL-NH(2)) were less active. An analogue of BLAST-2 with C-terminus in acid form and a pseudopeptide analogue of BLAST-2 with a methyleneamino Psi[CH(2)NH] peptide bond surrogate between residues L(3) and Y(4) were inactive. Corazonin elicited rapid, reversible and dose-dependent cardioacceleratory activity. When tested together with BLAST-1, corazonin overrode the cardioinhibitory effect of allatostatin. Our previous results had shown that high levels of allatostatin were maintained during the period of oothecal transport. This and the fact that physiological concentrations of allatostatins produce physiological levels of inhibition, suggest that allatostatins are involved in the modulation of cardiac rhythm in this cockroach.

Localization of allatostatin-immunoreactive material in the central nervous system, stomatogastric nervous system, and gut of the cockroach Blattella germanica.

Immunoreactivity against peptides of the allatostatin family having a typical YXFGL-NH2 C-terminus has been localized in different areas of the central nervous system, stomatogastric nervous system and gut of the cockroach Blattella germanica. In the protocerebrum, the most characteristic immunoreactive perikarya are situated in the lateral and median neurosecretory cell groups. Immunoreactive median neurosecretory cells send their axons around the circumesophageal connectives to form arborizations in the anterior neuropil of the tritocerebrum. A group of cells in the lateral aspect of the tritocerebrum project to the antennal lobes in the deutocerebrum, where immunoreactive arborizations can be seen in the periphery of individual glomeruli. Nerve terminals were shown in the corpora allata. These terminals come from perikarya situated in the lateral neurosecretory cells in the pars lateralis and in the subesophageal ganglion. Immunoreactive axons from median neurosecretory cells and from cells positioned in the anteriormost part of the tritocerebrum enter together in the stomatogastric nervous system and innervate foregut and midgut, especially the crop and the valve between the crop and the midgut. The hindgut is innervated by neurons whose perikarya are located in the last abdominal ganglion. Besides immunoreactivity in neurons, allatostatin-immunoreactive material is present in endocrine cells distributed within the whole midgut epithelium. Possible functions for these peptides according to their localization are discussed.

Identification, tissue localisation and physiological effect in vitro of a neuroendocrine peptide identical to a dipteran Leu-callatostatin in the codling moth Cydia pomonella (Tortricidae: Lepidoptera).

A neuroendocrine peptide of the Leu-callatostatin family, LPVYNFGL-NH2, has been isolated from tissue extracts of 5th instar larvae of the codling moth, Cydia pomonella (Lepidoptera). It is identical to a peptide previously isolated from the blowfly, Calliphora vomitoria (Diptera). The distribution of this peptide within the tissues of C. pomonella has been mapped by immunocytochemistry using antisera raised against LPVYNFGL-NH2. Midgut endocrine cells contain Leu-callatostatin immunoreactivity, as do several paired Leu-callatostatin neurones in the brain and ventral nerve cord. Within the visceral nervous system, the frontal ganglion contains four Leu-callatostatin neurones. Axons from these cells combine with others originating from neurones in the brain and project within the nervi cardiostomatogastrici to innervate the tissues of the foregut. In particular, the oesophageal valve has a prominent ring of Leu-callatostatin-immunoreactive fibres. The synthetic peptide, LPVYNFGL-NH2, has a potent reversible inhibitory effect in vitro on all visible forms of spontaneous contractile activity of the foregut, including closure of the oesophageal valve. Complete myoinhibition is observed at peptide concentrations from 10(-10 )to 10(-16) M. These results, in conjunction with the results of similar studies on cockroaches, crickets and flies, suggest that the Leu-callatostatins are a ubiquitous family of insect neuroendocrine peptides with an important role in the control of gut motility.

Lepidopteran peptides of the allatostatin superfamily.

Peptides of the allatostatin superfamily with the C-terminal amino acid sequence -YXFGL-NH2 have been isolated and identified from the lepidopterans, the codling moth, Cydia pomonella (Tortricidae) and the bollworm, Helicoverpa armigera (Noctuidae). The peptides, designated cydiastatins and helicostatins respectively, were monitored during purification with radioimmunoassays based on the callatostatins of the blowfly Calliphora vomitoria. The eight peptides from each of the two species appear to form an homologous series with four identical and three that differ by a single amino acid. This study demonstrates the ubiquitous nature of this family of peptides in insects.

Isolation and identification of multiple neuropeptides of the allatostatin superfamily in the shore crab Carcinus maenas.

20 neuropeptides belonging to the allatostatin superfamily were isolated from extracts of cerebral and thoracic ganglia of the shore crab Carcinus maenas. They were purified by HPLC, monitored by radioimmunoassay and identified by mass spectrometry and amino acid sequencing. The allatostatins are characterised by a common C-terminal pentapeptide sequence -YXFGL-NH2. Previously such peptides have only been reported from insects. In insects the variable post-tyrosyl residue is restricted to Ala, Asn, Asp, Gly or Ser. In C. maenas, however, there are only two types; thirteen of the peptides having a post-tyrosyl Ala and the other seven, a post-tyrosyl Ser. The crab peptides include the shortest allatostatins so far identified (YAFGL-NH2 and YSFGL-NH2) as well as the longest, a 27-residue peptide. The total of 20 peptides exceeds the highest number of allatostatins found in any of the insects investigated so far (14 in Periplaneta americana). It is of interest that, despite their clear homology, none of the peptides of C. maenas is identical to any of the more than 50 known insect allatostatins. The crab allatostatins show evidence of gene duplication and mutation that has resulted in several sub-groups with close structural similarities. For example, there are four heptapeptides with the common C-terminus -PYAFGL-NH2 that differ only at the N-terminal residue, which is either Glu, Asp, Asn or Ser. Other motifs, variously extended at the N-terminus, include -GPY(A/S)FGL-NH2 (three peptides), -DMY(A/S)FGL-NH2 (three peptides), and -GQY(A/S)FGL-NH2 (two peptides). Unique among the allatostatin superfamily, one of the crab peptides has a Tyr for Phe substitution at position three from the C-terminus (GGPYSYGL-NH2). Immunocytochemistry has provided clues to the functions of the allatostatins in crustaceans by showing their widespread presence in the central and stomatogastric nervous systems.

Identification of the dipteran Leu-callatostatin peptide family: the pattern of precursor processing revealed by isolation studies in Calliphora vomitoria.

Information from the Leu-callatostatin gene sequences of the blowflies Calliphora vomitoria and Lucilia cuprina was used to develop antisera specific for the variable post-tyrosyl amino-acid residues Ser, Ala and Asn of the common Leu-callatostatin C-terminal pentapeptide sequence -YXFGL-NH2. Radioimmunoassays based on these antisera were used to purify peptides from an extract of 40000 blowfly heads. Five neuropeptides of the Leu-callatostatin family were identified. Three have a seryl residue in the post-tyrosyl position. Two of these are octapeptides that differ only at the N-terminal residue; NRPYSFGL-NH2 and ARPYSFGL-NH2, whilst the third is the heptapeptide derived by N-terminal trimming; RPYSFGL-NH2. Two octapeptides in which X is Ala and Asn were also identified; VERYAFGL-NH2 and LPVYNFGL-NH2. The latter peptide is derived by processing at the internal dibasic site of a putative heneicosapeptide encoded by the DNA. These findings stress the necessity to have putative structures verified at the peptide level. Potent, reversible inhibitory effects on the spontaneous contractile activity of the blowfly rectum were recorded for ARPYSFGL-NH2 (monophasic dose-response curve with an IC50 = 10 fM) and for LPVYNFGL-NH2 (biphasic dose-response curve with IC50 values of approximately 1 fM and 1 nM). It is suggested that regulation of gut motility in insects, rather than an allatostatic function, may represent an ancestral and universal function of the allatostatins. One of the reasons for the large number of members of the Leu-callatostatin family appears to be in the provision of an integrated form of gut motility control, with different peptides controlling specific regions of the gut.

Allatostatic neuropeptides from the cockroach Blattella germanica (L.) (Dictyoptera, Blattellidae). Identification, immunolocalization and activity.

Four allatostatic neuropeptides were isolated from extracts of the brain of the cockroach Blattella germanica. The primary structures of these peptides were assigned as Leu-Tyr-Asp-Phe-Gly-Leu-NH2 (BLAST-1), Asp-Arg-Leu-Tyr-Ser-Phe-Gly-Leu-NH2 (BLAST-2), Ala-Gly-Ser-Asp-Gly-Arg-Leu-Tyr-Ser-Phe-Gly-Leu-NH2 (BLAST-3) and Ala-Pro-Ser-Ser-Ala-Gln-Arg-Leu-Tyr-Gly-Phe-Gly-Leu-NH2 (BLAST-4). Each of the peptides showed C-terminal amino acid sequence similarity to cockroach allatostatins and blowfly callatostatins. The four peptides inhibited in vitro juvenile hormone production by corpora allata from virgin females of B. germanica. Immunoreactivity against allatostatins was seen in the lateral neurosecretory neurons and in the axonal pathway leading to the corpora allata.

Insulin and IGF-I receptors and tyrosine kinase activity in carp ovaries: changes with reproductive cycle.

Insulin and insulin-like growth factor I (IGF-I) receptors from carp ovaries were semipurified with wheat germ agglutinin at different moments of the reproductive cycle and their binding characteristics and tyrosine kinase activity were studied. Specific receptors for insulin and IGF-I were found. IGF-I receptors presented higher binding (23.8 ± 1.5%), number of receptors (965 ± 20fm/mg) and affinity (KD 0.24 ± 0.03nM) than those shown for insulin receptors (4.1 ± 1%, 530 ± 85fm/mg and 0.85 ± 0.1nM, respectively). Insulin and IGF-I receptors have a tyrosine kinase activity which is not different from that found in muscle of the same species. Seasonal changes were found in binding, with maximum values for insulin and IGF-I reached at the end of pre-spawning period (June). However, while IGF-I binding was observed in all stages, insulin binding decreased in autumn and disappeared in winter, which suggests a different role for the two peptides in ovarian physiology.