cAMP-dependent protein kinase of Manduca sexta phosphorylates but does not activate the fat body triglyceride lipase

cAMP-dependent-protein kinase (PKA) is a central player of the adipokinetic signal that controls the mobilization of stored lipids in the fat body. Previous studies showed that adipokinetic hormone (AKH) rapidly activates PKA from the fat body of Manduca sexta (Arrese et al. (J. Lipid. Res. 40(3): 556)). As a part of our investigation on lipolysis in insects, here we report the purification and characterization of the catalytic subunit of PKA from the fat body of M. sexta and its role in the direct activation of the TG lipase in vitro. PKA was purified to apparent homogeneity and the identity of the protein was confirmed by MALDI-TOF and Western blot analysis. The enzyme showed a high affinity for Mg-ATP (Km = 39 microM) and Kemptide (Km = 31 microM) and was strongly inhibited by the PKA specific inhibitors PKI 5-24 and H89. Manduca sexta PKA only recognized serine residues as phosphate acceptor; theronine or tyrosine containing peptides were not phosphorylated. Purified fat body TG-lipase proved to be a good substrate of the purified kinase. However, phosphorylation of the lipase did not enhance the lipolytic activity of the enzyme in vitro. These results suggest that, besides lipase phosphorylation, the mechanism of AKH-induced activation of the lipolysis requires the involvement of other proteins and/or signals.     

Red pigment-concentrating hormone is not limited to crustaceans

A peptide that was previously assumed to occur exclusively in crustaceans is found in the corpora cardiaca of the stinkbug, Nezara viridula. The sequence of the peptide was deduced from the multiple MS(N) electrospray mass data as that of an octapeptide: pGlu-Ile/Leu-Asn-Phe-Ser-Pro-Gly-Trp amide. This peptide with Leu at position 2 is known as crustacean red pigment-concentrating hormone and code-named Panbo-RPCH. The ambiguity about the amino acid at position 2, Leu or Ile, was solved by isolating the peptide in a single-step by reversed-phase HPLC and establishing co-elution with authentic Panbo-RPCH but not with the Ile(2)-analog. When injected into stinkbugs, synthetic Panbo-RPCH elicited an increase of lipids in the haemolymph. Thus, it is assumed that Panbo-RPCH functions in the stinkbug as a lipid-mobilizing hormone.     

Isolation and immunochemical characterization of neuropeptides from the midgut of the Colorado potato beetleLeptinotarsa decemlineata

The midgut of the Colorado potato beetle showed endocrine cells immunopositive to monoclonals like MAC-18, MAC-3 and polyclonals to FMRF-amide and AKH-241. Extraction and HPLC-fractionation of the midgut extracts after partial purification and characterization showed immunopositive reaction to the monoclonals and also showed myotropic activity stimulating the potato beetle gut. Molecular mass of the two active peptides isolated were 22 and 26 kDa respectively. Both these peptides could be immunocytochemically demonstrated in the brain neurosecretory cells of the red cotton bug,Dysdercus cingulatus and the castor semilooper,Achaea janata as well.     

Locust flight activity as a model for hormonal regulation of lipid mobilization and transport

Flight activity of insects provides a fascinating yet relatively simple model system for studying the regulation of processes involved in energy metabolism. This is particularly highlighted during long-distance flight, for which the locust constitutes a long-standing favored model insect, which as one of the most infamous agricultural pests additionally has considerable economical importance. Remarkably many aspects and processes pivotal to our understanding of (neuro)hormonal regulation of lipid mobilization and transport during insect flight activity have been discovered in the locust; among which are the peptide adipokinetic hormones (AKHs), synthesized and stored by the neurosecretory cells of the corpus cardiacum, that regulate and integrate lipid (diacylglycerol) mobilization and transport, the functioning of the reversible conversions of lipoproteins (lipophorins) in the hemolymph during flight activity, revealing novel concepts for the transport of lipids in the circulatory system, and the structure and functioning of the exchangeable apolipopotein, apolipophorin III, which exhibits a dual capacity to exist in both lipid-bound and lipid-free states that is essential to these lipophorin conversions. Besides, the lipophorin receptor (LpR) was identified and characterized in the locust.In an integrative approach, this short review aims at highlighting the locust as an unrivalled model for studying (neuro)hormonal regulation of lipid mobilization and transport during insect flight activity, that additionally has offered a broad and profound research model for integrative physiology and biochemistry, and particularly focuses on recent developments in the concept of AKH-induced changes in the lipophorin system during locust flight, that deviates fundamentally from the lipoprotein-based transport of lipids in the circulation of mammals. Current studies in this field employing the locust as a model continue to attribute to its role as a favored model organism, but also reveal some disadvantages compared to model insects with a completely sequenced genome.     

Induced hyperlipaemia and immune challenge in locusts

Injections of immunogens, such as beta-1,3-glucan or lipopolysaccharide (LPS), bring about a marked hyperlipaemia with associated changes in lipophorins and apolipophorin-III in the haemolymph of Locusta migratoria. These changes are similar to those observed after injection of adipokinetic hormone (AKH). The possibility that endogenous AKH is released as part of the response to these immunogens is investigated using passive immunisation against AKH-I, and measurement of AKH-I titre in the haemolymph after injection of immunogens. The data presented show that, despite the similarity of the changes brought about by the presence of immunogens in the haemolymph to those brought about by AKH, there is no release of endogenous AKH after injection of laminarin or LPS. A direct effect of the immunogens on release of neutral lipids by the fat body cannot be demonstrated in vitro, and the mechanism by which hyperlipaemia is induced during immune challenge remains uncertain.     

Structures of two cockroach neuropeptides assigned by fast atom bombardment mass spectrometry

Amino acid sequences have been assigned to two cockroach neuropeptides (Glu-Val-Asn-Phe-Ser-Pro-Asn-Trp-NH₂, M I, and Glu-Leu-Thr-Phe-Thr-Pro-Asn-Trp-NH₂, M II) by application of fast atom bombardment mass spectrometry, including high resolution and linked scan (metastable) studies. The peptides show considerable homology with two other invertebrate neuropeptides, adipokinetic hormone (AKH, from a locust) and red pigment concentrating hormone (RPCH, from a prawn), whose fast atom bombardment spectra were also studied. M I and M II are thus members of a family of structurally-related invertebrate neuropeptides.     

Metabolism of Stored Reserves in Insect Fat Body: Hormonal Signal Transduction Implicated in Glycogen Mobilization and Biosynthesis of the Lipophorin System*

The mobilization of carbohydrate and lipid reserves from the insect fat body as fuels for migratory flight activity is controlled by adipokinetic hormone (AKH), of which in Locusta migratoria three different forms occur: AKH-I, -II and -III. In fat body in vitro, each AKH is capable of activating glycogen phosphorylase and of stimulating cAMP production, but only in the presence of extracellular Ca²⁺. The hormones stimulate both the influx and the efflux of Ca²⁺, the higher influx probably causing an increase in intracellular [Ca²⁺]. AKH enhances the production of inositol phosphates among which inositol 1,4,5-triphosphate may mediate the mobilization of Ca²⁺ from intracellular stores. Evidence is presented in favor of the occurrence of a capacitative calcium entry mechanism. Results suggest that transduction of the AKH signal occurs through stimulatory G protein-coupled receptor(s). A tentative model is presented for the interactions between the AKH signaling pathways in the locust fat body cell. AKH-induced lipid mobilization during flight requires the presence in the insect blood of high-density lipophorin (HDLp) particles and apolipophorin III (apoLp-III). Both protein components are synthesized in the fat body. In the locust, the two integral, nonexchangeable HDLp apolipophorins (apoLp-I and -II) were shown to originate from a common precursor; an mRNA of 10.3 kb seems to code for this precursor protein. The models proposed for lipophorin assembly and secretion in a number of insects are not in agreement. The exchangeable apoLp-III may occur in two or more isoforms; locust apoLp-III is secreted from the fat body as one of the two isoforms and in the hemolymph converted into the truncated second one. The rationale for this process is as yet unknown.     

EVIDENCE THAT ECDYSIS IN THE LARVAL COCKROACH,Periplaneta americana L. IS TRIGGERED BY AN INCREASE IN THE CONCENTRATION OF HEMOLYMPH SUGAR

Ecdysis in insects can be defined as shedding of the cuticle at the end of a larval stadium. This event can only occur after the peak titer of ecdysteroid in the hemolymph has returned to a low level. In the cockroach Periplaneta americana, ecdysis is strongly correlated with a rise in the concentration of trehalose and glucose in the hemolymph, leading to the idea that a causal relationship may exist between both events. The objective in this study was to determine if an increase in hemolymph sugar level would shorten the time to ecdysis in cockroach larvae with experimentally delayed ecdysis. The last larval stadium of P. americana averages 33.5 days but this increases significantly if the larva is injected with a small volume of saline. Injection of 10 μl of saline on day 20 and on four successive days lengthened the stadium by as much as 2 weeks. If, however, trehalose or glucose is incorporated into the saline, approximately 40% of the treated larvae undergo ecdysis at the same time as uninjected larvae. Injection of Peram‐AKH, the hypertrehalosemic hormone, also decreases the time for ecdysis to occur. This suggests that peak levels of ecdysteroid trigger the release of Peram‐AKH, which then leads to activation of trehalose synthesis. The results support the hypothesis that elevated hemolymph sugar is a contributing factor in the removal of ecdysteroid from the hemolymph.