The fat body plays major roles in the life of insects. It is a dynamic tissue involved in multiple metabolic functions. One of these functions is to store and release energy in response to the energy demands of the insect. Insects store energy reserves in the form of glycogen and triglycerides in the adipocytes, the main fat body cell. Insect adipocytes can store a great amount of lipid reserves as cytoplasmic lipid droplets. Lipid metabolism is essential for growth and reproduction and provides energy needed during extended nonfeeding periods. This review focuses on energy storage and release and summarizes current understanding of the mechanisms underlying these processes in insects. INSECT FAT BODY: AN OVERVIEWThe insect fat body plays an essential role in energy storage and utilization. It is the central storage depot for excess nutrients. In addition, it is an organ of great biosynthetic and metabolic activity (77). Fat body cells not only control the synthesis and utilization of energy reserves-fat and glycogen-but also synthesize most of the hemolymph proteins and circulating metabolites. Large amounts of relevant proteins, such as storage proteins used as an amino acid reservoir for morphogenesis, lipophorins responsible for the lipid transport in circulation, or vitellogenins for egg maturation, are secreted by the fat body (74). Most of the insect's intermediary metabolism takes place in this organ, including lipid and carbohydrate metabolism, protein synthesis, and amino acid and nitrogen metabolism. Some metabolic processes are stage specific such as the synthesis and secretion of storage proteins into the hemolymph that occur in the feeding larva or the synthesis of vitellogenin in adult insects.To perform multiple metabolic functions to fulfill the changing physiological needs of the insect during development, the fat body must be able to integrate signals from other organs. Many of these functions are hormonally regulated, and thus the fat body is the target organ of several hormones (47,107). At the same time, the fat body responds to the metabolic requirements of the organ itself. Therefore, several metabolic processes in the fat body must be tightly coupled to a number of metabolic pathways.Physiological systems to sense nutrient reserves are expected in all organisms, and in insects nutrient sensing itself appears to be the domain of the fat body (87). Studies of Drosophila melanogaster and, more recently, mosquitoes have shown that the fat body specifically expresses aminoacid transporters that function as nutrient sensors (14,61). The level of nutrient reserves accumulated in the fat body modulates several important aspects of the insect's life such as the rate of insect growth, the timing of metamorphosis, and egg development (87). The fat body coordinates insect growth with metamorphosis or reproduction by storing or releasing components central to these events. For example, the synthesis of vitellogenin in the fat body of Aedes aegypti female mosquitoes is NIH Public Access

Manduca sexta, known as the tobacco hornworm or Carolina sphinx moth, is a lepidopteran insect that is used extensively as a model system for research in insect biochemistry, physiology, neurobiology, development, and immunity. One important benefit of this species as an experimental model is its extremely large size, reaching more than 10 g in the larval stage. M. sexta larvae feed on solanaceous plants and thus must tolerate a substantial challenge from plant allelochemicals, including nicotine. We report the sequence and annotation of the M. sexta genome, and a survey of gene expression in various tissues and developmental stages. The Msex_1.0 genome assembly resulted in a total genome size of 419.4 Mbp. Repetitive sequences accounted for 25.8% of the assembled genome. The official gene set is comprised of 15,451 protein-coding genes, of which 2498 were manually curated. Extensive RNA-seq data from many tissues and developmental stages were used to improve gene models and for insights into gene expression patterns. Genome wide synteny analysis indicated a high level of macrosynteny in the Lepidoptera. Annotation and analyses were carried out for gene families involved in a wide spectrum of biological processes, including apoptosis, vacuole sorting, growth and development, structures of exoskeleton, egg shells, and muscle, vision, chemosensation, ion channels, signal transduction, neuropeptide signaling, neurotransmitter synthesis and transport, nicotine tolerance, lipid metabolism, and immunity. This genome sequence, annotation, and analysis provide an important new resource from a well-studied model insect species and will facilitate further biochemical and mechanistic experimental studies of many biological systems in insects.

BackgroundIn eukaryotes, microRNAs (miRNAs) have emerged as critical regulators of gene expression. The Silkworm (Bombyx mori L.) is one of the most suitable lepidopteran insects for studying the molecular aspects of metamorphosis because of its large size, availability of mutants and genome sequence. Besides, this insect also has been amply studied from a physiological and biochemical perspective. Deep sequencing of small RNAs isolated from different stages of silkworm is a powerful tool not only for measuring the changes in miRNA profile but also for discovering novel miRNAs.ResultsWe generated small RNA libraries from feeding larvae, spinning larvae, pupae and adults of B. mori and obtained ~2.5 million reads of 18-30 nt. Sequence analysis identified 14 novel and 101 conserved miRNAs. Most novel miRNAs are preferentially expressed in pupae, whereas more than 95% of the conserved miRNAs are dynamically regulated during different developmental stages. Remarkably, the miRNA-star (miR*) of four miRNAs are expressed at much higher levels than their corresponding miRNAs, and their expression profiles are distinct from their corresponding miRNA profiles during different developmental stages. Additionally, we detected two antisense miRNA loci (miR-263-S and miR-263-AS; miR-306-S and miR-306-AS) that are expressed in sense and antisense directions. Interestingly, miR-263 and miR-306 are preferentially and abundantly expressed in pupae and adults, respectively.ConclusionsWe identified 101 homologs of conserved miRNAs, 14 species-specific and two antisense miRNAs in the silkworm. Our results provided deeper insights into changes in conserved and novel miRNA and miRNA* accumulation during development.

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The hydrolysis of triglyceride (TG) stored in the lipid droplets of the insect fat body is under hormonal regulation by the adipokinetic hormone (AKH), which triggers a rapid activation cAMP-dependent kinase cascade (protein kinase A (PKA)). The role of phosphorylation on two components of the lipolytic process, the TG-lipase and the lipid droplet, was investigated in fat body adipocytes. The activity of purified TG-lipase determined using in vivo TG-radiolabeled lipid droplets was unaffected by the phosphorylation of the lipase. However, the activity of purified lipase was 2.4-fold higher against lipid droplets isolated from hormone-stimulated fat bodies than against lipid droplets isolated from unstimulated tissue. In vivo stimulation of lipolysis promotes a rapid phosphorylation of a lipid droplet protein with an apparent mass of 42-44 kDa. This protein was identified as "Lipid Storage Droplet Protein 1" (Lsdp1). In vivo phosphorylation of this protein reached a peak ϳ10 min after the injection of AKH. Supporting a role of Lsdp1 in lipolysis, maximum TG-lipase activity was also observed with lipid droplets isolated 10 min after hormonal stimulation. The activation of lipolysis was reconstituted in vitro using purified insect PKA and TG-lipase and lipid droplets. In vitro phosphorylation of lipid droplets catalyzed by PKA enhanced the phosphorylation of Lsdp1 and the lipolytic rate of the lipase, demonstrating a prominent role PKA and protein phosphorylation on the activation of the lipid droplets. AKH-induced changes in the properties of the substrate do not promote a tight association of the lipase with the lipid droplets. It is concluded that the lipolysis in fat body adipocytes is controlled by the activation of the lipid droplet. This activation is achieved by PKA-mediated phosphorylation of the lipid droplet. Lsdp1 is the main target of PKA, suggesting that this protein is a major player in the activation of lipolysis in insects.Insects rely on lipid reserves to survive during physiological non-feeding periods or to meet the energy requirements of developing eggs, flight, and starvation. The fat body is the principal site for storage of both glycogen and lipids. The fat body synthesizes most of the proteins found in the hemolymph while also serving as the main storage site of triglycerides (TG), 1 which constitute Ͼ90% fat body lipids. Therefore, functionally, the fat body accomplishes roles that in vertebrates are carried out by both liver and adipose tissue (1).The tobacco hornworm, Manduca sexta, feeds constantly, and the content of fat body TG increases continuously until the end of the larval development. During the larval period (ϳ20 days), the content of TG in the fat body increases from a few micrograms to ϳ80 mg (2). During subsequent development, the lipid reserves are used to sustain the life of the adult insect, which feeds occasionally (2-5). Due to these metabolic features, M. sexta represents an excellent model for studying the basic mechanisms involved in either the synthesis/deposition o...

Late embryogenesis abundant (LEA) proteins are members of a large group of hydrophilic, glycine-rich proteins found in plants, algae, fungi, and bacteria known collectively as hydrophilins that are preferentially expressed in response to dehydration or hyperosmotic stress. Group 2 LEA (dehydrins or responsive to abscisic acid) proteins are postulated to stabilize macromolecules against damage by freezing, dehydration, ionic, or osmotic stress. However, the structural and physicochemical properties of group 2 LEA proteins that account for such functions remain unknown. We have analyzed the structural properties of a recombinant form of a soybean (Glycine max) group 2 LEA (rGmDHN1). Differential scanning calorimetry of purified rGmDHN1 demonstrated that the protein does not display a cooperative unfolding transition upon heating. Ultraviolet absorption and circular dichroism spectroscopy revealed that the protein is in a largely hydrated and unstructured conformation in solution. However, ultraviolet absorption and circular dichroism measurements collected at different temperatures showed that the protein exists in equilibrium between two extended conformational states: unordered and left-handed extended helical or poly (l-proline)-type II structures. It is estimated that 27% of the residues of rGmDHN1 adopt or poly (l-proline)-type II-like helical conformation at 12°C. The content of extended helix gradually decreases to 15% as the temperature is increased to 80°C. Studies of the conformation of the protein in solution in the presence of liposomes, trifluoroethanol, and sodium dodecyl sulfate indicated that rGmDHN1 has a very low intrinsic ability to adopt ␣-helical structure and to interact with phospholipid bilayers through amphipathic ␣-helices. The ability of the protein to remain in a highly extended conformation at low temperatures could constitute the basis of the functional role of GmDHN1 in the prevention of freezing, desiccation, ionic, or osmotic stress-related damage to macromolecular structures. Group 2 LEA proteins or dehydrins or responsive to abscisic acid (RAB) proteins were originally identified as the "D-11" family of LEA proteins in developing cotton (Gossypium hirsutum) embryos (Baker et al., 1988;Dure et al., 1989;Hughes and Galau, 1989). Dehydrins appear to be ubiquitously expressed in gymnosperms (Jarvis et al., 1996;Richard et al., 2000) and angiosperms (Campbell and Close, 1997;Close, 1997). Immunological surveys have also detected dehydrin-related proteins in algae, yeast, and cyanobacteria (Close and Lammers, 1993; Campbell and Close, 1997;Close, 1997;Li et al., 1998;Mtwisha et al., 1998). Group 2 LEA proteins form a subset of evolutionarily conserved Gly-rich, hydrophilic proteins associated with adaptation to hyperosmotic conditions (Garay-Arroyo et al., 2000). Dehydrins are induced typically in maturing seeds or vegetative tissues following salinity, dehydration, cold, or freezing stress or abscisic acid (ABA) treatment (Close, 1996(Close, , 1997 Campbell and Close, 1997). Numero...

Surface plasmon resonance (SPR) spectroscopy can provide useful information regarding average structural properties of membrane films supported on planar solid substrates. Here we have used SPR spectroscopy for the first time to monitor the binding and activation of G-protein (transducin or Gt) by bovine rhodopsin incorporated into an egg phosphatidylcholine bilayer deposited on a silver film. Rhodopsin incorporation into the membrane, performed by dilution of a detergent solution of the protein, proceeds in a saturable manner. Before photolysis, the SPR data show that Gt binds tightly (Keq approximately equal to 60 nM) and with positive cooperativity to rhodopsin in the lipid layer to form a closely packed film. A simple multilayer model yields a calculated average thickness of about 57 A, in good agreement with the structure of Gt. The data also demonstrate that Gt binding saturates at a Gt/rhodopsin ratio of approximately 0.6. Moreover, upon visible light irradiation, characteristic changes occur in the SPR spectrum, which can be modeled by a 6 A increase in the average thickness of the lipid/protein film caused by formation of metarhodopsin II (MII). Upon subsequent addition of GTP, further SPR spectral changes are induced. These are interpreted as resulting from dissociation of the alpha-subunit of Gt, formation of new MII-Gt complexes, and possible conformational changes of Gt as a consequence of complex formation. The above results clearly demonstrate the ability of SPR spectroscopy to monitor interactions among the proteins associated with signal transduction in membrane-bound systems.

Group 1 late embryogenesis-abundant (LEA) proteins are a subset of hydrophilins that are postulated to play important roles in protecting plant macromolecules from damage during freezing, desiccation, or osmotic stress. To better understand the putative functional roles of group 1 LEA proteins, we analyzed the structure of a group 1 LEA protein from soybean (Glycine max). Differential scanning calorimetry of the purified, recombinant protein demonstrated that the protein assumed a largely unstructured state in solution. In the presence of trifluoroethanol (50% [w/v]), the protein acquired a 30% ␣-helical content, indicating that the polypeptide is highly restricted to adopt ␣-helical structures. In the presence of sodium dodecyl sulfate (1% [w/v]), 8% of the polypeptide chain adopted an ␣-helical structure. However, incubation with phospholipids showed no effect on the protein structure. Ultraviolet absorption and circular dichroism spectroscopy revealed that the protein existed in equilibrium between two conformational states. Ultraviolet absorption spectroscopy studies also showed that the protein became more hydrated upon heating. Furthermore, circular dichroism spectral measurements indicated that a minimum of 14% of amino acid residues existed in a solvent-exposed, left-handed extended helical or poly (l-proline)-type (PII) conformation at 20°C with the remainder of the protein being unstructured. The content of PII-like structure increased as temperature was lowered. We hypothesize that by favoring the adoption of PII structure, instead of the formation of ␣-helical or ␤-sheet structures, group 1 LEA proteins retain a high content of surface area available for interaction with the solvent. This feature could constitute the basis of a potential role of LEA proteins in preventing freezing, desiccation, or osmotic stress damage.Late embryogenesis-abundant (LEA) proteins accumulate to high concentrations in plant embryos during the latter stages of seed development before desiccation (Baker et al., 1988;Dure et al., 1989;Hughes and Galau, 1989). LEA proteins also accumulate in vegetative tissues exposed to exogenous abscisic acid, as well as dehydration, osmotic, and lowtemperature stress (Chandler and Robertson, 1994;Ingram and Bartels, 1996; Bray, 1997; Close, 1996 Close, , 1997Thomashow, 1998; Nylander et al., 2001). More than seven different groups of LEA proteins have been described and categorized by virtue of similarities in their deduced amino acid sequences (Baker et al., 1988;Dure et al., 1989). The majority of LEA proteins are highly hydrophilic and display a preponderance (e.g. Ala, Gly, Glu, and Thr) or lack (e.g. Trp and Cys) of certain amino acid residues (Dure, 1993a(Dure, , 1993b(Dure, , 1997. Thus, LEA proteins are part of a larger, evolutionarily conserved group of hydrophilic proteins termed "hydrophilins" involved in various adaptive responses to hyperosmotic conditions (Garay-Arroyo et al., 2000).Various functions have been proposed for different groups of LEA proteins ranging from wa...