The role of the crustacean octapeptide red pigment concentrating hormone (RPCH) in the control of crayfish retinal activity was explored. RPCH injection into intact animals resulted, after a latency of 10-30 min, in a dose-dependent enhancement of electroretinogram (ERG) amplitude lasting 60-120 min. RPCH was able to potentiate ERG amplitude in both light-adapted and dark-adapted animals. Following light-adaptation, responsiveness to RPCH was five times higher than following dark-adaptation. In conjunction with ERG enhancement, in light-adapted animals, RPCH injection elicited a dose-dependent retraction of distal retinal pigment, but did not affect proximal retinal pigment position. The effects of RPCH were blocked by a polyclonal antibody raised against a tyrosinated form of RPCH (A-tyr-RPCH). The antibody was also capable of partially blocking the nocturnal phase of the circadian rhythm of ERG amplitude and the darkness-induced retraction of distal retinal pigment. These results suggest that RPCH acts both on the retinal photoreceptors and on the distal pigment cells, playing a physiological role as a mediator of the effects induced by darkness and by the nocturnal phase of the circadian rhythm (Garfias, 1995).
The octapeptide red pigment-concentrating hormone is capable of eliciting the aggregation of intracellular pigment granules in distal retinal pigment cells of isolated retinas of the crayfish Procambarus clarkii (Girard). The final level and the time course of pigment aggregation are dose dependent within a range of 10-10 mol l-1 to 10-4 mol l-1. The effect of red pigment-concentrating hormone is prevented by previous incubation with an anti-red pigment-concentrating hormone antibody; however, application of the antibody after the onset of the red pigment-concentrating hormone effect, does not prevent its full development. A similar effect to that elicited by red pigment-concentrating hormone is induced by the calcium ionophores ionomycin and A-23187. Red pigment-concentrating hormone evokes entry of 45Ca2+ to retinal cells. However, the red pigment-concentrating hormone-induced pigment aggregation persists in the presence of the calcium channel blocker verapamil and in Ca2+-free solutions. Caffeine and thapsigargin, known to release calcium from intracellular stores, elicit distal pigment aggregation, while ryanodine and dantrolene, blockers of intracellular calcium release, as well as the intracellular calcium chelator bapta-AM suppress the effect of red pigment-concentrating hormone. These results suggest that red pigment-concentrating hormone elicits distal retinal pigment aggregation by increasing intracellular calcium concentration, acting via a dual mechanism: (1) promoting calcium entry, and (2) releasing intracellular calcium (Porras, 2001).
The primary structures of two neuropeptides, Tabanus atratus adipokinetic hormone (Taa-AKH) and Tabanus atratus hypotrehalosemic hormone (Taa-HoTH), from the corpora cardiaca of horse flies (Diptera: Tabanidae) have been determined. Amino acid sequences of Taa-AKH (less than Glu-Leu-Thr-Phe-Thr-Pro-Gly-Trp-NH2) and Taa-HoTH (less than Glu-Leu-Thr-Phe-Thr-Pro-Gly-Trp-Gly-Tyr-NH2) (where less than Glu = pyroglutamic acid) were determined by automated gas-phase Edman degradation of the peptides deblocked by pyroglutamate aminopeptidase and by fast atom bombardment mass spectrometry. The hormones were synthesized, and the natural and synthetic peptides exhibited identical chromatographic, spectroscopic, and biological properties. When assayed in adult face fly males, Taa-AKH and Taa-HoTH demonstrated hyperlipemic activity, in addition, Taa-HoTH also demonstrated a significant hypotrehalosemic activity (Jaffee, 1989).
A hypertrehalosaemic neuropeptide from the corpora cardiaca of the blowfly Phormia terraenovae has been isolated by reversed-phase h.p.l.c., and its primary structure was determined by pulsed-liquid phase sequencing employing Edman chemistry after enzymically deblocking the N-terminal pyroglutamate residue. The C-terminus was also blocked, as indicated by the lack of digestion when the peptide was incubated with carboxypeptidase A. The octapeptide has the sequence pGlu-Leu-Thr-Phe-Ser-Pro-Asp-Trp-NH2 and is clearly defined as a novel member of the RPCH/AKH (red-pigment-concentrating hormone/adipokinetic hormone) family of peptides. It is the first charged member of this family to be found. The synthetic peptide causes an increase in the haemolymph carbohydrate concentration in a dose-dependent fashion in blowflies and therefore is named 'Phormia terraenovae hypertrehalosaemic hormone' (Pht-HrTH). In addition, receptors in the fat-body of the American cockroach (Periplaneta americana) recognize the peptide, resulting in carbohydrate elevation in the blood. However, fat-body receptors of the migratory locust (Locusta migratoria) do not recognize this charged molecule, and thus no lipid mobilization is observed in this species (Gade, 1990).
The peptide hormone which controls activation of fat body glycogen phosphorylase in starving larvae of Manduca sexta was isolated from larval corpora cardiaca and sequenced by FAB tandem mass spectrometry. It was found to be identical with Manduca AKH. This, together with earlier observations, demonstrates that in M. sexta AKH controls glycogen phosphorylase activation in starving larvae while in adults it controls lipid mobilization during flight. Larval corpora cardiaca contain about 10 times less AKH than the corpora cardiaca of adults. The corpora cardiaca of M. sexta appear to contain only one AKH (Ziegler, 1990).
Hypertrehalosemic hormone (a carbohydrate-mobilizing neuroendocrine decapeptide) and starvation markedly increases levels of a cockroach (Blaberus discoidalis) fat body cytochrome P450 message. The gene represented by the cloned P450 cDNA has been named CYP4C1 (cytochrome P450 family 4, subfamily C, gene 1), a newly identified member of the ubiquitous cytochrome P450 monooxygenase gene superfamily. Blaberus CYP4C1 (511 amino acids, Mr = 58,485) has a hydrophobic NH2 terminus and a sequence near the COOH terminus that is homologous to the cysteine-containing heme-binding region definitive of cytochromes P450. The cockroach sequence is 32%-36% identical to mammalian family 4A and 4B enzymes. It contains a 13-residue sequence characteristic of family 4 but not other P450s. This study suggests that CYP4C1 is hormonally regulated in association with energy substrate mobilization and supports the idea that family 4 is an old and widespread gene family (Bradfield, 1991).
A simple preparation designed to screen and compare the central action of putative neuroactive agents in the moth Manduca sexta is described. This approach combines microinjections into the central nervous system with myograms recorded from a pair of spontaneously active mesothoracic muscles. Pressure injection of either octopamine or Manduca adipokinetic hormone (M-AKH) into the mesothoracic neuropile increases the monitored motor activity. Under the conditions used, the excitatory effects of M-AKH exceed those of the potent neuromodulator octopamine. This suggests that M-AKH plays a role in the central nervous system in addition to its known metabolic functions and supports recent evidence that neuropeptides in insects can be multifunctional (Milde, 1995).
Blaberus hypertrehalosemic hormone (Bld-HTH)-dependent glycogen phosphorylase activation was investigated using in vitro fat bodies from the cockroach, Blaberus discoidalis. Resting levels of active phosphorylase were decreased by the presence of trehalose and glucose. Phosphorylase activation was dose-responsive to Bld-HTH and increased ca. 3-fold over a range of 0.02 to 2 nM Bld-HTH. Maximum phosphorylase activation required only 5-min exposure to Bld-HTH; reversion to the inactive state began within 15 min after Bld-HTH removal and was completed by 60 min. Octopamine also activated phosphorylase but required 10(3)-fold higher concentrations than did Bld-HTH. Concentrations of Bld-HTH and octopamine that increased active phosphorylase did not elevate fat body cAMP levels, although a high concentration of octopamine increased tissue cAMP levels. cAMP did not increase phosphorylase activity, but Ca2+ was important for both Bld-HTH- and octopamine-dependent phosphorylase activation (Park, 1995).
Signaling mechanisms for Blaberus discoidalis hypertrehalosemic hormone (Bld-HrTH)-dependent glycogen phosphorylase activation were investigated in vitro using fat body of the tropical cockroach, B. discoidalis. Brief treatment of fat bodies with Bld-HrTH in the absence of extracellular Ca2+ showed a significant activation of phosphorylase. Although extracellular Ca2+ was required for a full activation of phosphorylase by Bld-HrTH, stimulation in the absence of extracellular Ca2+ suggested that intracellular Ca2+ was also involved in Bld-HrTH signal transduction. Thapsigargin and thimerosal mobilize Ca2+ from intracellular stores by different mechanisms, and both chemicals stimulated phosphorylase activities as effectively as a maximum dose of Bld-HrTH. Bld-HrTH likely induces intracellular Ca2+ release followed by extracellular Ca2+ entry across the plasma membrane. Inositol-1,4,5-trisphosphate (InsP3) levels were greatly increased by Bld HrTH in a time- and dose-dependent manner, suggesting that InsP3 might be a Ca(2+)-mobilizing intracellular second messenger for Bld-HrTH (Park, 1996).
The pathway for the adipokinetic hormone-stimulated synthesis of sn-1,2-diacylglycerols in the adult Manduca sexta fat body was studied. Adult fat body lipids were labeled by feeding 5th instar larvae either with labelled oleic acid or glycerol and after 32 days insects at the adult stage were used. This long-term prelabeling led to labeled fat body acylglycerols in which triacylglycerols comprised the main radioactive lipid component (95.5%), regardless of the radiolabeled compound used. Because the distribution of radioactivity among the lipid classes was very close to the mass distribution of the fat body lipid subspecies, it was concluded that homogeneous labeling of fat body lipids was obtained. After adipokinetic hormone treatment, an accumulation of radioactivity in the sn-1,2-diacylglycerol fraction was the only significant change found in the distribution of radioactivity among fat body lipids. The size of diacylglycerol pool increased 280% 60 min after adipokinetic hormone stimulation, whereas the fatty acid, monoacylglycerol and phosphatidic acid pool sizes remained constant. These results support the hypothesis that adipokinetic hormone-stimulated synthesis of sn-1,2-diacylglycerol in the fat body involves stereospecific hydrolysis of the triacylglycerol stores (Arrese, 1997).
The corpora cardiaca (CC) of the Italian race (including also the africanised variety) of the honeybee (Apis mellifera ligustica) contain approximately 3 pmol of a hypertrehalosaemic peptide. This peptide is identical in structure to the adipokinetic hormone (AKH) found in Manduca sexta, Mas-AKH. The CC of the dark European race of the honeybee (Apis mellifera carnica) contain no detectable Mas-AKH or any other adipokinetic/hypertrehalosaemic peptide. This is the first report of the occurrence of this peptide in a non-lepidopteran insect and of an intraspecific variation with regards to the presence or absence of a hypertrehalosaemic peptide in the CC of an insect. Extracts of A. m. ligustica CC elicit a strong adipokinetic/hypertrehalosaemic response when injected into crickets and cockroaches but extracts of A. m. carnica CC elicit no such responses when injected into crickets, cockroaches and butterflies. A weak hypertrehalosaemic response to injected Mas-AKH was observed in winter bees of both races, but there was no response in spring/summer bees. However, if a seasonal difference exists, it is at best minimal. Honeybees always have access to a more than adequate supply of high energy food in the form of nectar or honey stored in the hive. Thus, though A. m. ligustica CC contain a hypertrehalosaemic peptide, there is neither a glycogen-mobilising function of this hormone nor an adequate glycogen store in their fat body for its effective utilisation (Lorenz, 1999).
Stereospecific hydrolysis of stored triacylglycerol by a phosphorylatable triacylglycerol-lipase is the pathway for the adipokinetic hormone-stimulated synthesis of sn -1, 2-diacylglycerol in insect fat body. The current series of experiments were designed to determine whether cAMP and/or calcium are involved in the signal transduction pathway for adipokinetic hormone in the fat body. After adipokinetic hormone treatment, cAMP-dependent protein kinase activity in the fat body rapidly increased and reached a maximum after 20 min, suggesting that adipokinetic hormone causes an increase in cAMP. Forskolin, an adenylate cyclase activator, induced up to a 97% increase in the secretion of diacylglycerol from the fat body. 8Br-cAMP (a membrane-permeable analog of cAMP) produced a 40% increase in the hemolymph diacylglycerol content. Treatment with cholera toxin, which also stimulates adenylate cyclase, induced up to a 145% increase in diacylglycerol production. Chelation of extracellular calcium produced up to 70% inhibition of the adipokinetic hormone-dependent mobilization of lipids. Calcium-mobilizing agents, ionomycin and thapsigargin, greatly stimulated DG production by up to 130%. Finally, adipokinetic hormone caused a rapid increase of calcium uptake into the fat body. These findings indicate that the action of adipokinetic hormone in mobilizing lipids from the insect fat body involves both cAMP and calcium as intracellular messengers (Arrese, 1999).
Four locustatachykinins (LomTK I-IV) were identified in about equal amounts in extracts of corpora cardiaca of locusts, using reverse-phase high-performance liquid chromatography and radioimmunoassay with synthetic LomTK I-IV as standards. Brain extracts also contained the four isoforms in roughly equimolar concentrations. Retrograde tracing of the nervi corporis cardiaci II (NCC II) in vitro with Lucifer yellow in combination with LomTK immunocytochemistry revealed that about half of the secretomotor neurons in the lateral part of the protocerebrum projecting into the glandular lobe of the corpora cardiaca (CCG) contain LomTK-immunoreactive material. Since the four LomTKs are present in the CCG, these four or five neurons in each hemisphere are likely to contain colocalized LomTK I-IV. The role of two of the LomTKs in the regulation of the release of adipokinetic hormones (AKHs) from the adipokinetic cells in the CCG in the locust was investigated. Experiments performed in vitro showed that LomTK I and II induced release of AKH in a dose-dependent manner. These peptides also rapidly and transiently elevated the cyclic AMP-content of the CCG. The peak level of cyclic AMP occurred about 45 seconds after stimulation with LomTK. These results support the proposal that LomTKs are involved in controlling the release of the adipokinetic hormones and suggest that all LomTK isoforms may participate in this cyclic AMP-mediated event (Nassel, 1999).
Feeding effects on hypertrehalosemic hormone (HTH) transcript levels in corpora cardiaca (CC) of adult females of the cockroach, Blaberus discoidalis were measured using dot blot hybridization. HTH transcript levels were nearly doubled in CC from females withheld from food and water for ten days compared to CC from fed females. The increase in HTH-mRNA was a response to starvation, not dehydration, and reversed within 2 days after exposure to food. HTH-mRNA was elevated in CC from fed insects that had their recurrent nerve severed, but low fecal output by insects with severed nerves indicated that feeding and digestion were impaired. Thus, the increased HTH synthesis likely resulted from starvation rather than disruption of neural regulation. CC from starved females that were refed with either solutions or agar that contained glucose did not show down-regulation of HTH-mRNA. Likewise, injections of glucose or trehalose did not suppress HTH-mRNA levels in CC of starving insects. Down-regulation of the starvation-related increase in HTH-mRNA appears to be a response to consumption of a complex of nutrients and not to increased carbohydrates or mechanical aspects of feeding (Keeley, 1998).
Adipokinetic hormone (AKH)-producing cells in the corpus cardiacum of the insect Locusta migratoria represent a neuroendocrine system containing large quantities of stored secretory peptides. The question whether the release of AKHs from these cells induces a concomitant enhancement of their biosynthesis has been addressed. The effects of hormone release in vivo (by flight activity) and in vitro (using crustacean cardioactive peptide, locustamyoinhibiting peptide, and activation of protein kinase A and C) on the biosynthetic activity for AKHs were measured. The intracellular levels of prepro-AKH mRNAs, the intracellular levels of pro-AKHs, and the rate of synthesis of (pro-)AKHs were used as parameters for biosynthetic activity. The effectiveness of in vitro treatment was assessed from the amounts of AKHs released. Neither flight activity as the natural stimulus for AKH release, nor in vitro treatment with the regulatory peptides or signal transduction activators appeared to affect the biosynthetic activity for AKHs. This points to an absence of coupling between release and biosynthesis of AKHs. The strategy of the AKH-producing cells to cope with variations in secretory stimulation seems to rely on a pool of secretory material that is readily releasable and continuously replenished by a process of steady biosynthesis (Hawthoorn, 2001).
A new hypertrehalosaemic peptide [Tea-HrTH; pQLNFSTGWGG-NH(2)] was isolated from the corpora cardiaca (CC) of the sawfly Tenthredo arcuata. The hypertrehalosaemic peptides found in the CC of five Bombus species and the paper wasp Polistes fuscata were identical to the adipokinetic hormone II of the desert locust, Schistocerca gregaria (Scg-AKH-II). The hypertrehalosaemic peptides found in the yellowjacket Vespula vulgaris and the hornet Vespa crabro were identical to the adipokinetic hormone of the cricket, Gryllus bimaculatus (Grb-AKH). All species examined had a large storage crop which, when filled with honey, held up to one-third of their total body weight. Overwintering queens of P. fuscata had large stores of carbohydrates and lipids in the abdomen, and were able to survive months of fasting. Workers of Bombus hortorum (bumble-bee), Apis mellifera (honey-bee) and V. vulgaris had little or no fat body. These species could fly as long as sugar was present in their crops, but they stopped flying as the carbohydrates in the crop disappeared. There was no significant increase in the haemolymph carbohydrate titres after injections of CC extracts or corresponding synthetic peptides into workers of B. hortorum or into males and females of T. arcuata. There was a moderate increase in haemolymph carbohydrate titres when these peptides were injected into overwintering queens of P. fuscata and into workers of V. crabro, both with significant amounts of fat body. However, well-fed V. vulgaris workers, with very little fat body, also responded to their own hypertrehalosaemic peptide (Lorenz, 2001).
This report examines three aspects of adipokinetic hormone (AKH) involvement in migratory flight behavior in the grasshopper, Melanoplus sanguinipes. The titer of hemolymph AKH I during long-duration tethered flight was examined using radioimmunoassay (RIA) after narrow bore RP-HPLC. The hemolymph fraction containing AKH I was assayed using commercially available anti-Tyr1-AKH I serum. Titer determinations of hemolymph AKH were done at rest and after various periods of flight. The amount of AKH I released from the corpora cardiaca during flight was estimated. When resting levels of AKH I and II in corpora cardiaca (CC) of migrants and non-migrants were examined with HPLC, no significant differences in AKH levels were detected between non-migrants, animals that had flown for 1 h to identify them as migrants, and animals that had flown to exhaustion (i.e., voluntary cessation). CC levels of both AKH I and II were less in this species than in locusts. When the lipid mobilization in response to AKH I and II was compared in migrants (animals that had self-identified as migrants in a 1-h tethered flight test) and non-migrants (animals that would not perform a 1-h flight in a tethered flight test), the adipokinetic response to AKH I was greater in migrants than in non-migrants, possibly indicating differences in level of sensitivity or number of receptors in the target tissues. AKH II had little effect on hemolymph lipid levels in either flight group, and may not play a significant role in lipid mobilization in this species (Min, 2004).
Triglycerides (TG) stores build up in the insect fat body as lipid droplets at times of excess of food. The mobilization of fat body triglyceride (TG) is stimulated by adipokinetic hormones (AKH). The action of AKH involves a rapid activation of cAMP-dependent protein kinase (PKA). Recent in vitro studies have shown that PKA phosphorylates and activates the TG-lipase substrate, the lipid droplets. Conversely, purified TG-lipase from Manduca sexta fat body is phosphorylated by PKA in vitro but is not activated. This study was directed to learn whether or not AKH promotes a change in the state of phosphorylation of the lipase in vivo, and what are the relative contributions of cytosol and lipid droplets to the overall increase of lipolysis triggered by AKH. TG-lipase activity of fat body cytosols isolated from control and AKH-treated insects was determined against the native substrate, in vivo [3H]-TG radiolabeled lipid droplets, obtained from control and AKH-treated insects. The lipase activity of the system composed of AKH-cytosol and AKH-lipid droplets was 3.1-fold higher than that determined with control cytosol and lipid droplets. Evaluation of the role of AKH-induced changes in the lipid droplets on lipolysis showed that changes in the lipid droplets are responsible for 70% of the lipolytic response to AKH. The remaining 30% appears to be due to AKH-dependent changes in the cytosol. However, the phosphorylation level of the TG-lipase was unchanged by AKH, indicating that phosphorylation of the TG-lipase plays no role in the activation of lipolysis induced by AKH (Patel, 2006).
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