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ABSTRACT: Adipokinetic hormone (AKH) is a peptide hormone that regulates the nutritional state in insects by supporting the mobilization of lipids. In the present study, we manipulated AKH signaling to evaluate how metabolic state regulates feeding in an orthopteran insect, the two-spotted cricket, Gryllus bimaculatus. This was accomplished by RNA interference (RNAi) targeting the receptor gene for AKH [G. bimaculatus AKHR (GrybiAKHR)]. We found that the knockdown of GrybiAKHR by AKHR-double-stranded RNA treatment decreased the levels of 1,2-diacylglycerol and trehalose in the hemolymph, whereas it increased the level of triacylglycerol in the fat body. In addition, the knockdown of GrybiAKHR enhanced starvation resistance and increased food intake. Furthermore, direct observation of GrybiAKHR(RNAi) crickets revealed that the knockdown of GrybiAKHR increased feeding frequency but did not alter meal duration, whereas locomotor activity decreased. The increased frequency of feeding by GrybiAKHR(RNAi) crickets eventually resulted in an increase of food intake. These data demonstrate that the regulation of the metabolic state by AKH signaling affects feeding frequency, probably through nutritional control.
Endocrinology 05/2012; 153(7):3111-22. · 4.46 Impact Factor
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ABSTRACT: The cDNAs encoding allatotropin (AT) and allatotropin-like peptides (ATLPs) were isolated from the silkworm, Bombyx mori. Similar to those of the tobacco hornworm, Manduca sexta, four peptides (AT, ATLP1, ATLP2, and ATLP3) are present in three different variants generated by alternative splicing. RT-PCR analyses showed that these splice variants are expressed in the central nervous system with differing expression patterns in each ganglion. Immunohistochemistry using an anti-AT antibody confirmed that AT-expressing cells were located in these central nervous ganglia as well as in two large anterior cells of the frontal ganglia. Injection of synthetic AT and ATLP-1 into B. mori larvae increased the latency to feed, indicating that AT and ATLP might function in the regulation of feeding behavior in B. mori.
Peptides 01/2012; 34(1):98-105. · 2.43 Impact Factor
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ABSTRACT: In our previous report, we demonstrated the possibility that various regulatory neuropeptides influence feeding behavior in the silkworm, Bombyx mori. Among these feeding-related neuropeptides, short neuropeptide F (sNPF) exhibited feeding-accelerating activity when injected into B. mori larvae. Like other insect sNPFs, the deduced amino acid sequence of the cDNA encoding the sNPF precursor appears to produce multiple sNPF and sNPF-related peptides in B. mori. The presence of three sNPFs, sNPF-1, sNPF-2, and sNPF-3, in the brain of B. mori larvae was confirmed by direct MALDI-TOF mass spectrometric profiling. In addition, all three sNPFs are present in other larval ganglia. The presence of sNPF mRNA in the central nervous system (CNS) was also confirmed by Reverse transcription-polymerase chain reaction. Semi-quantitative analyses of sNPFs in the larval brain using matrix-assisted laser desorption ionization time-of-flight mass spectrometry further revealed that brain sNPF levels decrease in response to starvation, and that they recover with the resumption of feeding. These data suggest that sNPFs were depleted by the starvation process. Furthermore, food deprivation decreased the transcriptional levels of the sNPF receptor (BNGR-A10) in the brain and CNS, suggesting that the sNPF system is dependent on the feeding state of the insect and that the sNPF system may be linked to locomotor activity associated with foraging behavior. Since the injection of sNPFs accelerated the onset of feeding in B. mori larvae, we concluded that sNPFs are strongly related to feeding behavior. In addition, semi-quantitative MS analyses revealed that allatostatin, which is present in the larval brain, is also reduced in response to starvation, whereas the peptide level of Bommyosuppressin was not affected by different feeding states.
Frontiers in endocrinology. 01/2012; 3:3.
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ABSTRACT: We recently identified a novel feeding-modulating peptide, hemolymph major anionic peptide (HemaP), designated Bommo-HemaP (B-HemaP), from hemolymph of the silkworm Bombyx mori. B-HemaP has a unique biological activity in modulating the regular frequency of feeding motivation, which is accompanied by increased foraging behaviors. To confirm the conservation of the HemaP-regulated feeding mechanism in lepidopteran species, we purified and sequenced two candidate peptides from the hemolymph of larvae of the sweet potato hornworm Agrius convolvuli. Unlike B. mori, A. convolvuli had two forms of HemaP, which were designated Agrco-HemaP-1 (A-HemaP-1) and Agrco-HemaP-2 (A-HemaP-2). The amino acid sequence of A-HemaP-2 was identical with that of A-HemaP-1, except for O-glycosylation on the fifth amino acid, threonine, within the N-terminal region. The amino acid sequence of A-HemaP-1/A-HemaP-2 had only 32% identity with B-HemaP. Structural analysis revealed that the carbohydrate moiety of A-HemaP-2 was an α-GalNAc residue. Injection of A-HemaP-1, A-HemaP-2 and recombinant A-HemaP-1 (rA-HemaP-1) individually caused a significant increase in foraging behaviors in A. convolvuli larvae, and no significant differences were observed among these three A-HemaPs. The CD spectra of these three A-HemaPs were quite similar, and all had α-helix-rich secondary structures. Although A-HemaP-1 and B-HemaP did not exhibit cross-reactivity at any injection doses examined, HemaP might be a conserved molecule among lepidopteran species that can modulate feeding motivation through the fluctuation of peptide levels in hemolymph.
FEBS Journal 01/2012; 279(1):168-79. · 3.79 Impact Factor
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Toshiaki Fukushima,
Hiroshi Okajima,
Daisuke Yamanaka,
Miyako Ariga, Shinji Nagata,
Akihiro Ito,
Minoru Yoshida,
Tomoichiro Asano,
Kazuhiro Chida,
Fumihiko Hakuno,
Shin-Ichiro Takahashi
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ABSTRACT: Prolonged stimulation of FRTL-5 thyroid cells with cAMP-generating agents including thyroid-stimulating hormone (TSH) or cAMP analogues potentiates tyrosine phosphorylation of insulin receptor substrate (IRS)-2 triggered by insulin-like growth factor (IGF)-I, leading to enhancement of IGF-I-dependent proliferation. Because we identified HSP90 as an IRS-2-interacting protein, the roles of HSP90 in potentiation of IGF signals through IRS-2 were investigated. We found that prolonged dibutyryl cAMP treatment induced serine/threonine phosphorylation of IRS-2. Using a specific inhibitor of HSP90 chaperone activity, geldanamycin, or small interfering RNA against HSP90, we showed that HSP90 mediates cAMP-induced serine/threonine phosphorylation of IRS-2. Furthermore, inhibition of HSP90 by geldanamycin during dibutyryl cAMP pretreatment of cells for 24h suppressed cAMP-dependent potentiation of tyrosine phosphorylation of IRS-2 induced by IGF-I. Taking together, we conclude that HSP90 interacting with IRS-2 mediates cAMP-dependent serine/threonine phosphorylation of IRS-2 via its chaperone activity, leading to potentiation of tyrosine phosphorylation of IRS-2 induced by IGF-I.
Molecular and Cellular Endocrinology 07/2011; 344(1-2):81-9. · 4.19 Impact Factor
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ABSTRACT: Sterols in silkworm larvae were analyzed. Cholesterol was predominantly detected in all tissues examined. Dietary phytosterols and desmosterol, a putative biosynthetic intermediate from phytosterols to cholesterol, were also detected, indicating that imperfect intestinal conversion from phytosterols to cholesterol influences the sterol composition in larval tissues.
Bioscience Biotechnology and Biochemistry 05/2011; 75(5):1003-5. · 1.28 Impact Factor
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ABSTRACT: In insects, especially phytophagous insects, feeding behavior occurs at a regular frequency. Although a number of physiological studies have revealed various causal factors leading to feeding behavior in insects, little has been demonstrated regarding the regulatory mechanisms underlying insect feeding behavior. To confirm the presence of an endocrinological regulatory mechanism in feeding behavior, we tested the effects of several biologically active peptides on silkworm, Bombyx mori larvae feeding behaviors. To evaluate the effects of the biologically active peptides, we measured the period of latency to the first bite following sample injection into starved Bombyx larvae. Of the chemically synthesized peptides tested, myosuppressin exhibited a prolonged latency, indicating that myosuppressin is a possible inhibitory peptide in Bombyx larvae. In contrast, injections of tachykinin and short neuropeptide F, which are members of the structurally related RF-amide peptide family, had a shorter latency period, indicating that these two peptides are possible stimulatory peptides. In addition, the present study suggests that this bioassay will be advantageous for screening for peptides that regulate insect feeding behavior.
General and Comparative Endocrinology 03/2011; 172(1):90-5. · 3.27 Impact Factor
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ABSTRACT: Crustacean hyperglycemic hormone (CHH), a member of a neuropeptide family present only in arthropods, plays a pivotal role in the modulation of hemolymph glucose levels, molting, reproduction, and the stress response. Although it has been determined that hepatopancreas and muscle are the major tissues in which CHH regulates hyperglycemic activity, the molecular mechanism by which CHH regulates carbohydrate metabolism remains unclear. In this study, we analyzed the mRNA expression levels of enzymes involved in glycogen metabolism and gluconeogenesis in order to determine how CHH regulates hemolymph glucose levels. We first cloned cDNAs encoding four carbohydrate metabolism-related enzymes from the kuruma prawn, Marsupenaeus japonicus, glycogen phosphorylase (MjGP), glycogen synthase (MjGS), fructose 1,6-bisphosphatase (MjFBPase), and phosphoenolpyruvate carboxykinase (MjPEPCK). RT-PCR analysis showed that eyestalk ablation remarkably decreased MjGP and increased MjGS transcript levels in the hepatopancreas, but not in muscle. Considering the fact that various eyestalk factors, including MIH, are removed by eyestalk ablation, these results indicate that after eyestalk ablation the metabolic state proceeds towards glycogen accumulation in the specific tissues related to molting. In contrast, MjFBPase and MjPEPCK transcript levels were not significantly changed by eyestalk ablation, indicating that CHH and other eyestalk-derived factors might not induce gluconeogenesis. Quantitative real-time PCR analysis showed that exposure of hepatopancreas to recombinant CHH significantly changed the expression levels of MjGP and MjGS, but not MjFBPase and MjPEPCK. Collectively, these results indicate that CHH is involved in glycogen metabolism in hepatopancreas.
General and Comparative Endocrinology 03/2011; 172(2):293-304. · 3.27 Impact Factor
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ABSTRACT: It has been thought that phosphorus in biominerals made of amorphous calcium carbonate (ACC) might be related to ACC formation, but no such phosphorus-containing compounds have ever been identified. Crustaceans use ACC biominerals in exoskeleton and gastroliths so that they will have easy access to calcium carbonate inside the body before and after molting. We have identified phosphoenolpyruvate and 3-phosphoglycerate, intermediates of the glycolytic pathway, in exoskeleton and gastroliths and found them important for stabilizing ACC.
Nature Chemical Biology 02/2011; 7(4):197-9. · 14.69 Impact Factor
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ABSTRACT: Phytophagous insects do not constantly chew their diets; most of their time is spent in a non-feeding quiescent state even though they live on or around their diets. Following starvation, phytophagous insect larvae exhibit enhanced foraging behaviors such as nibbling and walking similar to the sequential behavior that occurs prior to each meal. Although extensive physiological studies have revealed regularly occurring feeding behaviors in phytophagous insects, little has been elucidated regarding the mechanism at the molecular level. Here, we report identification and characterization of a novel 62-amino acid peptide, designated as hemolymph major anionic peptide (HemaP), from the hemolymph of Bombyx mori larvae that induces foraging behaviors. The endogenous HemaP levels are significantly increased by diet deprivation, whereas refeeding after starvation returns them to basal levels. In larvae fed ad libitum, hemolymph HemaP levels fluctuate according to the feeding cycle, indicating that locomotor-associated feeding behaviors of B. mori larvae are initiated when HemaP levels exceed an unidentified threshold. Furthermore, administration of exogenous HemaP mimics the starvation-experienced state by affecting dopamine levels in the suboesophageal ganglion, which coordinates neck and mandible movements. These data strongly suggest that fluctuation of hemolymph HemaP levels modulates the regularly occurring feeding-motivated behavior in B. mori by triggering feeding initiation.
Journal of Biological Chemistry 12/2010; 286(9):7161-70. · 4.77 Impact Factor
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ABSTRACT: Molting in crustaceans is induced by ecdysteroids as in insects. The ecdysteroid titre in hemolymph is negatively regulated by molt-inhibiting hormone (MIH) that inhibits the secretion of ecdysteroids from the Y-organ, an ecdysteroid-producing gland of crustaceans, whereas little is known about the molecular mechanism of inhibition by MIH. Recently, the Halloween genes encoding cytochrome P450 monooxygenases were characterized as the steroidogenic enzymes in insects. To elucidate whether the ecdysteroidogenesis in the Y-organ is regulated by molt-inhibiting hormone (MIH), we analyzed the expression level of an orthologue of a member of the Halloween genes, phantom (Cyp306a1, phm), in the Y-organ of a decapod crustacean, Marsupenaeus japonicus. A cDNA encoding phm (Mj-phm) was cloned by degenerate PCR and 5'- and 3'-RACEs. The deduced amino acid sequence of Mj-phm showed about 40% identity to those of insect phm. The six motif sequences and the four substrate recognition sites were well conserved between Mj-PHM and other PHM. RT-PCR showed the specific expression of Mj-phm mRNA in the Y-organ. In addition, quantitative real-time PCR verified that the expression level of Mj-phm was significantly increased at the pre-molt stage and decreased after ecdysis. Furthermore, exposure of the Y-organ to MIH significantly decreased the Mj-phm expression level in vitro. These results indicate that the transcription of Mj-phm in the Y-organ may be regulated by the inhibitory mechanism of MIH of M. japonicus, which involves the consequent negative regulation of ecdysteroidogenesis at the transcriptional level.
Archives of Insect Biochemistry and Physiology 10/2009; 72(4):220-33. · 1.36 Impact Factor
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ABSTRACT: To address the signaling pathway of crustacean hyperglycemic hormone (CHH) in the kuruma prawn Marsupenaeus japonicus, we measured the intracellular cAMP and cGMP levels in the hepatopancreas, one of the major target tissues of CHH. Removal of CHH-producing cells by bilateral eyestalk ablation decreased intracellular cGMP levels, while little effect on cAMP was observed in the tissue. Similarly, exposure to recombinant CHH significantly increased the cGMP level of the hepatopancreas in vitro, while little change was observed in cAMP. These results suggest that cGMP, rather than cAMP, mediates the CHH signal in hepatopancreas of M. japonicus.
Annals of the New York Academy of Sciences 05/2009; 1163:478-80. · 3.15 Impact Factor
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ABSTRACT: In several phytophagous insects, feeding behavior occurs regularly. Recently, we demonstrated that feeding behavior in larvae of the silkworm Bombyx mori had a regular frequency. To address the control of the feeding cycle in B. mori, we aimed to characterize factors influencing feeding initiation and termination. Injection of extracts of the midgut, foregut, and fat body into starved Bombyx larvae delayed the initiation of feeding. This result indicates the presence in these tissues of factors capable of decreasing the likelihood of feeding initiation.
Annals of the New York Academy of Sciences 05/2009; 1163:481-3. · 3.15 Impact Factor
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ABSTRACT: Crustacean hyperglycemic hormone (CHH) not only plays an important role in the modulation of hemolymph glucose level but also functions in other biological events including molting, reproduction and stress response. Of the six CHHs characterized in Marsupenaeus japonicus, an expression system for recombinant Pej-SGP-VII (rPej-SGP-VII-amide) has not yet been established. Here, we established a procedure using a Nus-tag for solubilization, thereby soluble and biologically active rPej-SGP-VII-amide could successfully be obtained by a simpler procedure than previous ones used for producing other recombinant Pej-SGPs (Pej-SGP-I, III and IV). It was found that rPej-SGP-VII-amide thus obtained had the correct arrangement of intramolecular disulfide bonds and helix-rich secondary structure. The established expression system for rPej-SGP-VII-amide may be applicable for the preparation of other recombinant CHHs.
Peptides 09/2008; 30(3):507-17. · 2.43 Impact Factor
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ABSTRACT: Two cDNAs encoding EcR (MjEcR) and RXR (MjRXR) were cloned and sequenced from the kuruma prawn Marsupenaeus japonicus using PCR techniques. The amino acid sequence of MjEcR was similar to that of known EcR especially in the ligand binding domain (LBD) of insect EcR. The DNA binding domain of MjRXR showed higher homology with that of insect USP (>90% identity) than vertebrate RXR ( approximately 85% identity), while LBD of MjRXR is more homologous with that of vertebrate RXR ( approximately 65% identity) than that of insect USP (30-60% identity). The transcripts of MjEcR and MjRXR were expressed in all tissues examined and in particular, highly in Y-organ and heart and in ovary and heart, respectively. Quantitative real-time PCR analyses revealed that the expression level of MjEcR in hepatopancreas and thoracic muscle increased from intermolt to premolt stages. The analyses also showed that the expressions of MjEcR and MjRXR were regulated in a tissue-specific manner. No significant changes were observed in reproductive organs throughout the molting stages, and MjRXR was expressed much more than MjEcR at all stages. These data suggest that MjRXR mediates a certain hormonal signal related to reproduction.
Comparative Biochemistry and Physiology Part B Biochemistry and Molecular Biology 10/2007; 148(2):139-50. · 1.92 Impact Factor
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ABSTRACT: Since insects are unable to biosynthesize sterols de novo, sterols must be obtained from dietary sources. Although it has been reported that beta-sitosterol is crucial for larval growth in the silkworm, Bombyx mori, little has been investigated concerning the dietary selection of sterols by Bombyx larvae. Here, we demonstrate that Bombyx larvae have the following sterol preference: beta-sitosterol > ergosterol > cholesterol = stigmasterol. Interestingly, Bombyx larvae preferred ergosterol, an inhibitory sterol on larval growth, indicating that sterol selection following first contact of the diet with the mouth part might be different from the sterol recognition mechanism present in sterol metabolism.
Bioscience Biotechnology and Biochemistry 12/2006; 70(12):3094-8. · 1.28 Impact Factor
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ABSTRACT: Continuous observations of larvae of the silkworm, Bombyx mori, revealed that feeding occurred at regular intervals throughout larval development. To investigate possible factors influencing meal-timing, the behaviours of diet-deprived Bombyx larvae were also analysed. Diet-deprivation resulted in longer durations of the first meals after diet replacement, but did not affect feeding patterns. Furthermore, long-term diet-deprivation promoted wandering behaviour and a consequent delay in feeding after diet replacement. Under diet-deprivation conditions, meal-starts appeared to be inducible by defecation and physical stimulation. However, stimulation-induced meal-starts were dependent on the time elapsed since the larvae's previous meals. Provided that more than 1h had elapsed since their previous meals, larvae could be induced to feed by defecation and tapping. At less than 1h post-meal, larvae were less likely to begin feeding after defecation or physical stimulation. Activated locomotions such as wandering and feeding were observed in the long-term diet-deprived larvae only after diet blocks were replaced, while long-term diet-deprived larvae did not show activated locomotion during the absence of diet blocks. Collectively, these data suggest that a combination of elevated locomotion activity and the presence of diet may be necessary for the initiation of feeding in diet-deprived larvae.
Journal of Insect Physiology 09/2006; 52(8):807-15. · 2.24 Impact Factor
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ABSTRACT: In a previous paper, we reported the cloning of a cDNA encoding a putative receptor, Pgdr, from the prothoracic gland of the silkworm, Bombyx mori. Few studies concerning the orthologous cDNA of Pgdr in mammals, a growth hormone-inducible transmembrane protein (Ghitm) that encodes a putative receptor, have been performed. Analysis of the distribution of Ghitm expression revealed ubiquitous expression in mouse embryo and adult tissues, as well as mammalian cell lines. The pattern of Ghitm expression suggested that once Ghitm mRNA was expressed in the putative brain region of mouse embryo, Ghitm-expressing cells spread ubiquitously throughout all tissues during embryonic development. In addition, Western blot analyses demonstrated that cleavage of the N-terminal portion in GHITM appears to regulate the expression level, suggesting that cleavage is essential for the proper expression of GHITM.
Biochemical and Biophysical Research Communications 04/2006; 341(1):13-8. · 2.48 Impact Factor
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ABSTRACT: A cDNA encoding a novel heptahelical receptor from the prothoracic glands of the silkworm, Bombyx mori was cloned and sequenced during screening of a prothoracicotropic hormone (PTTH) receptor. Orthologs of this receptor are found not only in insects, but also in the vertebrates. In B. mori, ubiquitous expression of the mRNA was observed in the larva. Also, a higher expression level in the prothoracic glands was observed before molting and metamorphosis and was impaired after pupal molting. But, further analysis is required to confirm whether this receptor cDNA encodes the PTTH receptor.
Bioscience Biotechnology and Biochemistry 03/2006; 70(2):554-8. · 1.28 Impact Factor
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ABSTRACT: The insect molting hormone, ecdysone, induces a series of events critical for ecdysis, beginning with the formation of a complex between ecdysteroid receptor (EcR) and its partner protein, ultraspiracle (Usp). In the present study, we cloned cDNAs encoding EcR and Usp by RT-PCR and 5'-/3'-RACE from the common cutworm, Spodoptera litura, a destructive agricultural pest insect in East Asia. Hexahistidine-tagged recombinant EcR and Usp of S. litura were expressed in E. coli, and the proteins, after refolding, exhibited the specific binding to an ecdysone analogue in vitro.
Annals of the New York Academy of Sciences 05/2005; 1040:417-9. · 3.15 Impact Factor
