Insect Fat Body Research Articles

Regional and functional differentiation in the fat body of pharaoh's ant queens, Monomorium pharaonis (L.)

The insect fat body is generally described as a uniform tissue with multiple functions, but we have found evidence of cell differentiation in the Monomorium fat body.We show that the fat body of a mature egg-laying pharaoh's ant queen is a result of a preceding remodeling of cell material comprising at least 11 different fat cell types, located at specific positions in the head, alitrunk (thorax) and gaster (abdomen). The cell types are classified based on their position, histochemistry, ultrastructure, and immunoreactivity for vitellogenin/vitellin. Some of these cells are primordial cells present at emergence, others invade the histolysing flight muscle tissue, and still others disappear during the maturation process. Only one type, the subepidermal fat cell of the gaster, is active in vitellogenin synthesis and is the only cell type in close association with oenocytes. Although only this type produces vitellogenin, our material indicates that most fat cell types are essential to support egg production. In some queens vitellogenin was found to form crystals in ventral vitellogenin-producing fat cells. This indicates an imbalance between vitellogenin production in the fat cells and uptake in the oocytes, which is probably related to a cyclic regulation of egg production.

Comparative Analysis of the Binding of Antibodies Prepared against the Insect Spodoptera exigua and against the Mycopathogen Nomuraea rileyi

Polyclonal antibodies were produced in mice against Spodoptera exigua (beet armyworm) larval hemolymph and hemocytes and against cell wall surfaces of hyphal bodies and hyphae of the entomopathogenic hyphomycete Nomuraea rileyi. In addition to exhibiting strong activity against their original antigenic substrates, all of the antibodies cross-react extensively with other substrates. The hemolymph antibody binds to hemocytes and vice versa, and both antibodies cross-react to the insect fat body basement membrane (extracellular matrix (ECM) and to N. rileyi and Beauveria bassiana (another entomopathogenic fungus) cell wall surfaces (ECM). Likewise, the anti-fungal antibodies cross-react with S. exigua hemolymph and hemocytes, especially the granules that may contain ECM components, and with fat body basement membrane. These cross-reactivities are specific as indicated by negative controls in the microscopy and Western blotting assays. Parallel labeling experiments using Con A suggest that the reactive epitopes contain mannose; however, none of the antibodies bind to mannose residues of nonentomopathogenic Candida albicans or Saccharomyces cerevisiae yeast cells. Thus, these cross-reactivities suggest that the host mimicry expressed by surface components of entomopathogenic fungi represents an important pathogenic determinant.

Direct manipulation of insect reproduction by agents of parasite origin

Reproductive output of female Tenebrio molitor beetles is reduced upon infection with metacestodes of the rat tapeworm, Hymenolepis diminuta . We are using this as a model to investigate the adaptive significance of parasite-induced curtailment of insect reproduction. Production of the yolk protein vitellogenin (Vg) in the insect fat body is significantly reduced both in vitro and in vivo by metacestodes. Synthesis can be measured by using [14C]L–leucine incorporation, followed by immunoprecipitation. In this paper we demonstrate that a significant decrease in [14C]Vg can be produced by an acetic acid extract of the parasite. Conclusive evidence is presented that the active component(s) originate from the metacestodes: an extract of parasites grown entirely axenically has similar deleterious effects. The developmental stage of the metacestode is important: immature (stage I to II) parasites had greater capacity to suppress Vg synthesis than mature ones (stage V to VI). Examination of the chemical nature of the effector molecule(s) revealed that acetic–acid–extractable, boiling–resistant, pronase–sensitive agents in the molecular mass range 10 to 50 kDa reduced Vg synthesis by 47.4%. These data suggest that metacestodes produce a modulator molecule that directly affects insect vitellogenesis and, therefore, that reduction of host fitness may confer a selective advantage upon the parasite.

Calcium and cAMP are second messengers in the adipokinetic hormone-induced lipolysis of triacylglycerols in Manduca sexta fat body

We have previously shown that 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 (0.1 μm), 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. Our 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, E. L., M. T. Flowers, J. L. Gazard, and M. A. Wells. Calcium and cAMP are second messengers in the adipokinetic hormone-induced lipolysis of triacylglycerols in Manduca sexta fat body. J. Lipid Res. 1999. 40: 556–564.

Three Isoforms of a Hepatocyte Nuclear Factor-4 Transcription Factor with Tissue- and Stage-specific Expression in the Adult Mosquito

We cloned three isoforms of hepatocyte nuclear factor-4 (HNF-4) from the mosquito Aedes aegypti, designated AaHNF-4a, AaHNF-4b, and AaHNF-4c. AaHNF-4a and AaHNF-4b are typical members of the HNF-4 subfamily of nuclear receptors with high amino acid conservation. They differ in N-terminal regions and exhibit distinct developmental profiles in the female mosquito fat body, a metabolic tissue functionally analogous to the vertebrate liver. The AaHNF-4b mRNA is predominant during the previtellogenic and vitellogenic phases, while the AaHNF-4a mRNA is predominant during the termination phase of vitellogenesis, coinciding with the onset of lipogenesis. The third isoform, AaHNF-4c, lacks part of the A/B and the entire C (DNA-binding) domains. The AaHNF-4c transcript found in the fat body during the termination of vitellogenesis may serve as a transcriptional inhibitor. Both AaHNF-4a and AaHNF-4b bind to the cognate DNA recognition site in electrophoretic mobility shift assay. Dimerization of AaHNF-4c with other mosquito HNF-4 isoforms or with mammalian HNF-4 prevents binding to the HNF-4 response element. In transfected human 293T cells, AaHNF-4c significantly reduced the transactivating effect of the human HNF-4alpha1 on the apolipoprotein CIII promoter. Electrophoretic mobility shift assay confirmed the presence of HNF-4 binding sites upstream of A. aegypti vg and vcp, two yolk protein genes expressed in the female mosquito fat body during vitellogenesis. Therefore, HNF-4, an important regulator of liver-specific genes, plays a critical role in the insect fat body.

Multiple interactions between insect lipoproteins and fat body cells: extracellular trapping and endocytic trafficking

The binding and internalization of a circulating insect lipoprotein, high density lipophorin (HDLp), by insect fat body cells was studied at the electron-microscopic level using ultrasmall gold-labeled HDLp and DiI-labeled HDLp, which were visualized by silver enhancement and diaminobenzidine photoconversion, respectively. Internalization of HDLp seems to conflict with the selective process by which the lipids are transported between HDLp and fat body cells. The pathway followed by the internalized lipoproteins was investigated. In addition, the localizations of HDLp in fat body cells of young and older adult locusts were compared because of the previously reported agerelated differences in distribution of cell-associated and internalized HDLp. In the present study, internalized labeled HDLp was observed in early endosomes, late endosomes, and putative lysosomes. In older adults, these labeled structures were much less abundant than in young adults. Moreover, in these animals, the labeled endosomal/lysosomal vesicles were located close to the plasma membranes. A more intense labeling was observed in the extracellular matrix in older adults compared to young adults. In both developmental stages, an apparent accumulation of labeled HDLp was found in extracellular spaces. We propose that this entrapment of HDLp may be essential for selective lipid transport between HDLp and fat body cells.—Dantuma, N. P., M. A. P. Pijnenburg, J. H. B. Diederen, and D. J. Van der Horst. Multiple interactions between insect lipoproteins and fat body cells: extracellular trapping and endocytic trafficking.

The phospholipase C signaling pathway in locust fat body is activated via G q and not affected by cAMP

Crosstalk between signal transduction pathways provides a complex intracellular avenue for fine tuning of hormone-induced signals. Over the last few years, we have studied the signaling mechanisms of three locust adipokinetic hormones (AKHs), which control mobilization of energy reserves from insect fat body as fuels for flight and transduce their signals via adenylyl cyclase- and phospholipase C- (PLC) dependent pathways. In this study, we examine possible crosstalk between these signaling routes. We show that cAMP does not affect basal and AKH-stimulated inositol phosphate (InsP n) production. Incubation of fat body with aluminium fluoride, an activator of G proteins, increased InsP n levels by 77%, whereas cholera toxin and pertussis toxin were ineffective. This implies that fat body PLC is not activated by Gβγ, but possibly by G qα. The involvement of this G protein in AKH signaling was demonstrated by our observation that the GPAntagonist-2A, which antagonizes G q, attenuated glycogen phosphorylase activation by AKH-I. As plasma membrane Ca 2+ channels may constitute another target for cAMP-mediated modulation, we studied the type of channels involved in AKH signaling using a variety of L-, N- and T-type Ca 2+ channel inhibitors. None of these blocked AKH-induced glycogen phosphorylase activation, suggesting that voltage-dependent Ca 2+ channels do not mediate AKH-induced Ca 2+ influx.

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.

Glucogenesis in an insect, Manduca sexta L., estimated from the 13C isotopomer distribution in trehalose synthesized from [1,3- 13C 2]glycerol

Glucogenesis from [3- 13C]alanine and [1,3- 13C 2]glycerol was demonstrated in the insect Manduca sexta by examining the 13C enrichment of trehalose, a non-reducing disaccharide of glucose synthesized in the insect fat body and released into the blood or hemolymph. In insects maintained on a low carbohydrate diet, trehalose synthesized from [3- 13C]alanine was selectively enriched at C1 and C6, and C2 and C5. The 13C-labelling pattern indicated the carboxylation of [3- 13C]pyruvate, formed by transamination of the [3- 13C]alanine followed by randomization of the label at the fumarate step of the tricarboxylic acid cycle and glucose synthesis via the gluconeogenic pathway. 13C enrichment of trehalose was absent in similarly maintained insect larvae administered 3-mercaptopicolinic acid, an inhibitor of hepatic phosphoenolpyruvate carboxykinase. Insects on the low carbohydrate diet also synthesized trehalose from [1,3- 13C 2]glycerol. 13C multiplets were observed in trehalose C3 and C4 demonstrating the synthesis of three 13C enriched glucose isotopomers from the 13C-labelled glycerol. The relative contributions of 13C-labelled glycerol and unlabelled 3 carbon substrates to the synthesis of the 13C enriched trehalose isotopomers were determined from the multiplet structure at C3, and calculation of minimal rates of glucogenesis were based on the 13C enrichment of C4. The C4/C3 13C enrichment ratio in trehalose synthesized from [1,3- 13C 2]glycerol was close to unity, and total glucogenesis was calculated after estimation of the expected contribution of unlabelled trehalose synthesis from 3 carbon substrates by comparison of the ratio of unlabelled and labelled contributions to the 13C enriched trehalose isotopomers with the 13C enrichment of [1,3- 13C 2]glycerol-3-phosphate. The estimated total rates of glucogenesis varied from 0.33 to 2.80 μmol glucose/g fresh weight/h. The blood sugar level of M. sexta was also highly variable. Although the potential importance of glucogenesis from 3 carbon substrates to the maintenance of blood sugar was not established by the present investigation, insects maintained on the low carbohydrate diet had similar blood trehalose levels to those previously reported by others for insects maintained on a natural food.

Analysis of the mosquito lysosomal asparticp protease gene: An insect housekeeping gene with fat body-enhanced expression

In the fat body of insects with cyclic egg maturation, lysosomes play a critical role in the termination of vitellogenesis by selectively degrading the secretory machinery involved in the massive production of yolk protein precursors. To investigate this fat body-specific lysosomal activity in the mosquito, a cathepsin D-like aspartic protease (LAP) was previously purified and its cDNA cloned. Here we report the isolation of the AaLAP gene from an Aedes aegypti genomic library. The transcribed region of the gene is comprised of five exons, spanning 1904 base pairs. Restriction fragment length polymorphism (RFLP) and genomic clone analyses show this gene to be single copy and polymorphic. Primer extension analysis revealed two putative transcription start sites (TSS). The extension products corresponding to the distal and proximal TSSs were present in both pre- and vitellogenic fat bodies, suggesting that both TSSs are involved in housekeeping as well as tissue-specific expression of this gene. TATA box-like and arthropod initiator sequences, hallmarks of regulated genes, were present near each putative TSS. Several sequences resembling binding sites for liver- and fat body-specific transcription factors were identified within 1 kb upstream and downstream of the gene. Significantly, direct binding for the C/EBP and GATA families of transcription factors was demonstrated in vitro by electrophoretic mobility shift assays (EMSA). Three sequences located upstream of AaLAP resembled the Drosophila melanogaster yolk protein fat body enhancer ( DmYp FBE). Potential hormone-response elements were also recognized in the gene; however, they did not bind the mosquito ecdysteroid receptor/Ultraspiracle heterodimer in EMSA experiments, indicating that these sequences may interact with different nuclear receptors.

Inhibition of Neuronal High Voltage-activated Calcium Channels by Insect Peptides: a Comparison with the Actions of ω-Conotoxin GVIA

The whole cell variant of the patch clamp technique was used to investigate the actions of two novel insect peptides on high voltage-activated Ca 2+ currents in cultured dorsal root ganglion (DRG) neurones. The insect peptides (PMP-D2 and PMP-C) were isolated originally from insect brains and fat bodies, and have been found to have similar three-dimensional structures to the N-type Ca 2+ channel inhibitor ω-conotoxin GVIA (ω-CgTx GVIA). High voltage-activated Ca 2+ currents were activated from a holding potential of −90 mV by depolarizing step commands to 0 mV. Extracellular application of synthetic PMP-D2 or PMP-C (1 μM) attenuated high voltage-activated Ca 2+ currents. The effects of PMP-C were strongly dependent on the frequency of current activation, but inhibition was apparent and reached a steady state after 20 steps when currents were evoked for 30 msec at 0.1 Hz. The actions of the two insect peptides overlapped both with each other and with ω-CgTx GVIA, suggesting that N-type Ca 2+ current was predominantly sensitive to these peptides. Low voltage-activated T-type current and 1,4-dihydropyridine sensitive L-type Ca 2+ currents were insensitive to 1 μM PMP-D2 and PMP-C, which indicates a degree of selectivity. The presence of a fucose group on PMP-C abolished the ability of this peptide to attenuate high voltage-activated Ca 2+ currents, which may reflect a mechanism by which peptide function could be regulated in insects. The electrophysiological data are supported by studies on 45Ca 2+ influx into rat cerebrocortical synaptosomes. Both PMP-D2 (10 μM), PMP-C (10 μM) and ω-CgTx GVIA (1 μM) attenuated a proportion of 45Ca 2+ influx into the synaptosomes, but additive effects of these peptides were not observed. We conclude that these naturally occurring peptides obtained from invertebrate preparations have inhibitory effects on N-type Ca 2+ channels. Although the peptides have related three-dimensional structures, they have distinct amino acid sequences and appear to have different mechanisms of action to produce inhibition of mammalian neuronal high voltage-activated Ca 2+ channels. © 1997 Elsevier Science Ltd. All rights reserved.

Insect adipokinetic hormone stimulates inositol phosphate metabolism: roles for both Ins(1,4,5) P3 and Ins(1,3,4,5) P4 in signal transduction?

Adipokinetic hormones (AKHs) control the mobilization of energy reserves from the insect fat body as fuels for flight activity. As a part of our investigations on AKH signal transduction, we demonstrate in this study that the inositol lipid cycle may be involved in the action of AKH-I on fat body of the migratory locust. We show that [ 3H]inositol is incorporated into fat body phosphoinositides in vitro, whose hydrolysis leads to the formation of the following inositol phosphates (InsPs): Ins(1 and/or 3) P, Ins(4) P, Ins(1,3) P 2, Ins(1,4) P 2, Ins(3,4) P 2, Ins(1,3,4) P 3, Ins(1,4,5) P 3 and Ins(1,3,4,5) P 4. AKH stimulates the formation of these isomers, eliciting an increase in radioactivity of total Ins Ps already after 1 min. Mass measurements show that Ins(1,4,5)P 3 levels are substantially enhanced by AKH, which is indicative of hormonal activation of phospholipase C. In cell-free tissue preparations, Ins(1,4,5) P 3 is metabolized through dephosphorylation as well as further phosphorylation. Ins(1,3,4,5) P 4 is dephosphorylated primarily to Ins(1,3,4) P 3, although the a bility for its reconversion to Ins(1,4,5) P 3 suggests that in vivo Ins(1,3,4,5) P 4 may function as rapidly mobilizable pool for Ins(1,4,5) P 3 generation. Metabolic pathways for the conversion of InsPs to inositol in the locust fat body are proposed.

Lipid transfer from insect fat body to lipophorin: comparison between a mosquito triacylglycerol-rich lipophorin and a sphinx moth diacylglycerol-rich lipophorin

Two insect lipoproteins, triacylglycerol-rich Aedes aegypti lipophorin and diacylglycerol-rich Manduca sexta lipophorin, were compared in their ability to load neutral lipid from fat body. When fat body of M. sexta was incubated in vitro with [3H]oleic acid, all radiolabeled fatty acids were esterified, predominantly to triacylglycerol. In A. aegypti fat body, however, half of the label remained as free fatty acids. When A. aegypti fat body was radiolabeled with [3H]glycerol, most of the radiolabel was incorporated in triacylglycerol. When either A. aegypti or M. sexta lipophorin was incubated with A. aegypti fat body, labeled with [3H]oleic acid, both lipophorins incorporated mainly radiolabeled free fatty acids, while almost no radiolabeled glycerides were transferred. When the same experiment was performed with A. aegypti fat body, radiolabeled with [3H]glycerol, very little transfer of radiolabeled glycerides was detected. In contrast, when either M. sexta or A. aegypti lipophorin was incubated with M. sexta fat body, both lipophorins incorporated neutral lipids, predominantly diacyglycerol. A. aegypti lipophorin incorporated half the amount of radiolabeled lipid, compared to M. sexta lipophorin. Lipophorins from both species were treated with triacylglycerol lipase of the yeast Candida cylindracea. Although this lipase readily delipidated M. sexta HDLp, it was not able to remove triacylglycerol from A. aegypti HDLp. The data presented suggest that, under the conditions used, lipid transfer from fat body to lipophorin in A. aegypti is not as efficient as in M. sexta.

Synthesis of sn-1,2-diacylglycerols by monoacylglycerol acyltransferase from Manduca sexta fat body.

The pathway for the synthesis of sn-1,2-diacylglycerol stimulated by the action of adipokinetic hormone (AKH) in the insect fat body is unknown. Previous results from this laboratory suggested that the hydrolysis of stored triacylglycerol to sn-2-monoacylglycerol followed by the stereospecific acylation of sn-2-monoacylglycerol catalyzed by a monoacylglycerol-acyltransferase (MGAT) could be the major route of AKH-stimulated sn-1,2-diacylglycerol synthesis. Thus, MGAT might represent a key enzyme of this pathway. In this study we characterized the MGAT activity from the Manduca sexta fat body. The activity, which was assayed by acylation of 2-monoolein using radioactive labeled palmitoyl-CoA, was found to be primarily a microsomal enzyme. The products of the acylation of 2-monoolein were 1,2-diacylglycerol (40-50%), 1,3-diacylglycerol (20-30%), and triacylglycerol (30-40%). The presence of triacylglycerol as a product revealed the presence of diacylglycerol-acyltransferase activity in the fat body microsomes. The pH optimum of MGAT activity was 7.0, and the dependence of the activity on the concentration of 2-monoolein showed saturation kinetics. An endogenous MGAT activity, which represented 20% of the maximal activity observed with added substrate, was detected. Optimal concentrations of palmitoyl-CoA ranged between 0.10-0.20 mM. The specific activity of MGAT, measured under optimal conditions, was about 0.6 nmol DG formed/min-mg protein. MGAT activity was greatest with 2-monoolein, and lower activity was observed when a saturated 2-monoacylglycerol was employed. The activity observed with sn-1-monoacylglycerol was lower than that observed with sn-2-monoacylglycerol. AKH did not stimulate MGAT activity, suggesting that either the enzyme is not under hormonal regulation or the monoacylglycerol pathway is not involved in the AKH-stimulated production of sn-1,2-diacylglycerol in the M. sexta fat body.

Development and characterization of insect cell lines

With the wide availability of insect cell culture media, it can generally be considered a routine process to develop new cell lines. Exceptions to this statement do exist, of course. Difficulties may arise when attempting to culture a specific cell type. For example, while there are a few cell lines from insect fat body and at least one from the midgut, it may not be possible to obtain cell lines from these tissues from all insect species due to terminal differentiation and other factors. Also, researchers have desired cell lines from certain species, such as the honey bee, for which no success has been obtained. As in the early days of tissue culture, it is difficult to discern why negative results occur. However, as more is learned about the physiology and nutrition of various insects and tissues, we may get clues which will help solve these questions.The remaining chapters in this book will provide the reader with exciting uses for insect cell culture. As I mentioned earlier, the baculovirus expression vector system has provided a stimulus to the field of insect cell culture not seen previously.

Plant defensins: novel antimicrobial peptides as components of the host defense system.

Various mechanisms to fend off microbial invaders have been devised by all living organisms, including microorganisms themselves. The most sophisticated of these mechanisms relies on the synthesis of immunoglobulins directed against specific microbial targets. However, immunoglobulin-based immunity operates only in a relatively minor subset of living species, namely the higher vertebrates. A much more ancient and widespread defense strategy involves the production of small peptides that exert antimicrobial properties. As products of single genes, antimicrobial peptides can be synthesized in a swift and flexible way, and because of their small size they can be produced by the host with a minimal input of energy and biomass. Wellknown examples of antimicrobial peptides are the cecropins that accumulate in the hemolymph of many invertebrates in response to injury or infection (reviewed by Boman and Hultmark, 1987) and the magainins that are secreted by glands in the skin of amphibians (reviewed by Bevins and Zasloff, 1990). Cecropins and magainins are small (20-40 residues) basic peptides displaying an amphipathic a-helical structure that can integrate in microbial membranes to form ion channels (Duclohier, 1994). Another class of antimicrobial peptides is formed by the Cys-rich peptides, which in contrast to cecropins and magainins, have a complex cystine-stabilized three-dimensional folding pattern often involving antiparallel ,3-sheets. Defensins are one class among the numerous types of Cys-rich antimicrobial peptides, which differ in length, number of cystine, bonds, or folding pattern (reviewed by Boman, 1995). Insect (34-43 residues, three disulfide bridges) are, like cecropins, produced in a pathogeninducible manner by the insect fat body and secreted in the hemolymph (reviewed by Hoffmann and Hetru, 1992). Mammalian (29-34 amino acids, three disulfide bridges) are produced by various specialized cells in the mammalian body (reviewed by Lehrer et al., 1993; Ganz and Lehrer, 1994). For example, they are very abundant in granules of phagocytic blood cells. These granules fuse with phagocytosis vesicles containing microorganisms, where the are thought to contribute, together with other antimicrobial proteins and active oxygen species, to killing of the engulfed microorganisms. Defensins are also secreted by epithelial cells of the intestines and airways, where they may help maintain the normal microbial flora in a steady state. In addition, the expression of in the airway epithelium has been shown to be up-regulated after exposure to bacterial lipopolysaccharides (Diamond et al., 1993). The importance of in innate immunity of humans is underscored by the observation that certain disorders characterized by recurrent infections are associated with a lack of in blood phagocytes (Ganz et al., 1988). Moreover, transposon mutants of a pathogenic Salmonella strain known to infect and grow inside phagocytes simultaneously lost their resistance to (and other antimicrobial peptides) and their virulence (Groisman et al., 1992). Recently, we characterized a novel class of peptides whose structural and functional properties resemble those of insect and mammalian defensins. Hence, we termed this family of peptides plant defensins (Terras et al., 1995).

Signal transduction of adipokinetic hormones involves Ca 2+ fluxes and depends on extracellular Ca 2+ to potentiate cAMP-induced activation of glycogen phosphorylase

Adipokinetic hormone (AKH)-induced mobilization of insect fat body glycogen occurs through activation of glycogen phosphorylase. In the migratory locust, signal transduction of AKH-I, -II and -III has been shown to involve the formation of cAMP. In the present study, we show that both the elevation of fat body CAMP levels and the activation of phosphorylase by the three AKHs in vitro depend on the presence of extracellular Ca 2+; in the absence of Ca 2+ in the medium, no phosphorylase activation occurs, whereas a concentration of at least 1.5 mM Ca 2+ in the medium is required for maximal activation by each of the hormones. Furthermore, we show that AKH-I, -II and -III increase the influx of extracellular calcium into the fat body, as well as the efflux of cytosolic calcium from the fat body into the medium within 1 min of incubation. Although the time courses of their effects and the maximal responses to massive doses (40 nM) of the three hormones do not differ, AKH-III induces the highest increase in Ca 2+ efflux when applied in a physiological dose (4 nM). No difference in the levels of Ca 2+ influx induced by 4 nM of the hormones was observed. Quantitative analysis of the data suggests that the AKH-induced influx is larger than the efflux, implying a net rise in the fat body Ca 2+ concentration.

Regional and Functional Differentiation in the Insect Fat Body

Regional and Functional Differentiation in the Insect Fat Body

Regional and Functional Differentiation in the Insect Fact Body

Insect fat body historically has been considered a single tissue with multiple and diverse metabolic functions. Recent findings indicate that functionally distinct regions of fat body exist in some insects and that these regions often have unique morphologies. Regionally differentiated fat bodies in Lepidoptera have been associated with the segregation of synthetic and storage activities. The loss of biosynthetic capacity for storage proteins at the beginning of metamorphosis is the consequence of the histolysis of a larval specific histotype, whereas the histotype that accumulates storage proteins persists into the adult stage. In Diptera, functional histotypes are arranged along the anteriorposterior axis and between the larval and adult stages. Based on these models. we predict that the use of immunological and molecular probes will lead to the identification of regionally and functionally differentiated fat body histotypes in many insects.

Insect immunity. A transgenic analysis in Drosophila defines several functional domains in the diptericin promoter.

Diptericins are antibacterial polypeptides which are strongly induced in the fat body and blood cells of dipteran insects in response to septic injury. The promoter of the single-copy, intronless diptericin gene of Drosophila contains several nucleotide sequences homologous to mammalian cis-regulatory motifs involved in the control of acute phase response genes. Extending our previous studies on the expression of the diptericin gene, we now report a quantitative analysis of the contribution of various putative regulatory elements to the bacterial inducibility of this gene, based on the generation of 60 transgenic fly lines carrying different elements fused to a reporter gene. Our data definitively identify two Kappa B-related motifs in the proximal promoter as the sites conferring inducibility and tissue-specific expression to the diptericin gene. These motifs alone, however, mediate only minimal levels of expression. Additional proximal regulatory elements are necessary to attain some 20% of the full response and we suspect a role for sequences homologous to mammalian IL6 response elements and interferon-gamma responsive sites in this up-regulation. The transgenic experiments also reveal the existence of a distal regulatory element located upstream of -0.6 kb which increases the level of expression by a factor of five.