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Perilipins (PLINs) are proteins that associate with lipid droplets (LDs) and play roles in the control of triglycerides (TG) metabolism. Two types of PLINs - 1 and 2- occur in insects. Following previous work on MsPLIN1A (a 42 kDa protein formerly called MsLsd1), here we report a new PLIN1 isoform, MsPLIN1B. MsPLIN1B cDNA was cloned and the 1835bp cDNA contains an ORF encoding a 47.9 kDa protein whose expression was confirmed by mass spectrometry . Alternative transcripts A and B, which differ in the alternative use of exon 1, were the most abundant PLIN1 transcripts in the fat body. These transcripts encode nearly identical proteins except that the B isoform contains 59 additional residues in its amino terminus . No conserved domain was identified in the extra region of MsPLIN1B. The novel PLIN1 isoform is found in lepidopteran species. In Manduca , PLIN1B was expressed only in the 5th instar larva and its levels correlated with fat storage . Furthermore, PLIN1B levels increased with the fat content of the diet in insects of the same age confirming a direct relationship between PLIN1B and TG storage irrespective of development. The nutritional status impacted PLIN1B levels, which decreased in starvation and increased with subsequent re-feeding. Altogether data support a link between PLIN1B and TG storage in caterpillars prior to pupation . The combined findings suggest distinct roles for PLIN1A, PLIN1B and PLIN2. MsPLIN1A abundance correlates with mobilization of TG stores, MsPLIN2 with the synthesis of new LDs and MsPLIN1B abundance correlates with high levels of TG storage and large LD sizes at the end of the last feeding period.

Cellular triglycerides (TG) are stored in cytosolic lipid droplets (LDs). Perilipins (PLIN) are a group of LD-proteins that play important roles in the assembly and transport of LDs and in TG metabolism. Two members of the PLIN family are found in insects (PLIN1 & 2 or Lsd1 & 2). We have cloned and expressed Manduca sexta PLIN2 (MsPLIN2), and studied developmental and nutritional changes in the expression of PLIN2. Nutritional changes induced fast alterations in PLIN2 mRNA and protein levels in fat body and midgut of the feeding larvae. The relationship observed between PLIN2 expression and TG synthesis in both larval fat body and midgut suggests that PLIN2 is needed when tissues are accumulating TG. However, when the fat body was storing TG at maximal capacity, MsPLIN2 levels declined. This unexpected finding suggests the occurrence of alternative mechanism/s to shield TG from the action of lipases in M. sexta LDs. In addition, it implies that the cellular level of lipid storage could be modulating MsPLIN2 expression and/or degradation. The study also confirmed that MsPLIN2 was most abundant in the adult fat body, which is characterized by a high rate of TG hydrolysis and lipid mobilization. Whether MsPLIN2 is directly involved in lipolysis and/or the secretion of lipids in the fat body of adult of M. sexta is unknown at this time. Nonetheless, the coexistence of high PLIN2 and lipolysis levels suggests a complex role for MsPLIN2. Altogether, we found that MsPLIN2 is needed when the synthesis of glycerides, DG and TG, is active even if the insect is accumulating or consuming TG.

DmCatD, a cathepsin D-like peptidase of the hematophagous insect Dipetalogaster maxima, is synthesized by the fat body and the ovary and functions as yolk protein precursor. Functionally, DmCatD is involved in vitellin proteolysis. In this work, we purified and sequenced DmCatD, performed bioinformatic analyses and investigated the events involved in its targeting and storage in developing oocytes. By ion exchange and gel filtration chromatography, DmCatD was purified from egg homogenates and its identity was confirmed by mass spectrometry. Approximately 73% of the full-length transcript was sequenced. The phylogeny indicated that DmCatD has features which suggest its distancing from “classical” cathepsins D. Bioinformatic analyses using a chimeric construct were employed to predict post-translational modifications. Structural modeling showed that DmCatD exhibited the expected folding for this type of enzyme, and an active site with conserved architecture. The interaction between DmCatD and lipophorin in the hemolymph was demonstrated by co-immunoprecipitation. Colocalization of both proteins in developing oocyte membranes and yolk bodies was detected by immunofluorescence. Docking assays favoring the interaction DmCatD-lipophorin were carried out after modeling lipophorin of a related triatomine species. Our results suggest that lipophorin acts as a carrier for DmCatD to facilitate its further internalization by the oocytes. The mechanisms involved in the uptake of peptidases within the oocytes of insects have not been reported. This is the first experimental work supporting the interaction between cathepsin D and lipophorin in an insect species, enabling us to propose a pathway for its targeting and storage in developing oocytes. Fil: Leyria, Jimena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; Argentina Fil: Fruttero, Leonardo Luis. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; Argentina. Pontificia Universidade Católica do Rio Grande do Sul; Brasil. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina Fil: Ligabue Braun, Rodrigo. Universidade Federal do Rio Grande do Sul; Brasil Fil: Defferrari, Marina S.. University of Toronto; Canadá Fil: Arrese, Estela L.. Oklahoma State University; Estados Unidos Fil: Soulages, José L.. Oklahoma State University; Estados Unidos Fil: Settembrini, Beatriz Patricia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Museo Argentino de Ciencias Naturales “Bernardino Rivadavia”; Argentina Fil: Carlini, Célia Regina R S. Pontificia Universidade Católica do Rio Grande do Sul; Brasil Fil: Canavoso, Lilian Etelvina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; Argentina

Fatty acids stored as triglycerides (TG) in the fat body serve as precursor in multiple processes including energy production and synthesis of cellular components. Mobilization of fatty acids from TG depends on the action of lipases. The fat body triglyceride lipase from Manduca sexta, MsTGL, is the only insect lipase that has been purified and characterized, so far. A TGL cDNA from M. sexta fat body encoding a 649 amino acid protein was cloned and its identity confirmed by mass spectrometry and Edman sequencing data of the purified protein. The protein sequence has conserved domains and residues of potential importance for the function and regulation of TGL activity. The expression of TGL and the lipase activity of fat body homogenates were studied in several developmental stages of M. sexta. TG-hydrolase activity of fat body increased as larva grew to the last instar and, then, decreased to minimal levels during pupa stage. Lipase activity was progressively restored in adult insects and reached maximum values at this stage. The fat body lipase activity from adult insects, 1-2 day after emergence, was 9-fold higher than that from 2 to 3 days old 5th-instar larvae. A good correlation was found between the abundance of TGL protein and the lipase activity of fat body homogenates. This correlation and the expression pattern of TGL throughout development are consistent with the notion that TGL is the main fat body TG lipase of M. sexta.

Lipid storage protein 2 (Lsd 2) is a conserved insect protein that belongs to the small PAT family of proteins. PAT proteins are found associated to the lipid droplets of adipocytes and play significant roles in the regulation of triacylglycerides metabolism. Here we describe the expression and purification of Lsd2, its reconstitution in lipoprotein particles, the location of putative lipid binding sites and its secondary structure. This study provides the starting point for future studies on the mechanism of function of Lsd2. The similarities and differences between Lsd1 and Lsd2, the only PAT proteins found in insects, are discussed.

Cathelicidins, a group of cationic peptides found in leukocytes and epithelial cells, play a central role in the early innate immune defense against infection. Although these host defense peptides have been reported in several mammalian species, including primates, no cathelicidins have been identified in carnivores. Here we report the cloning, tissue expression and biological activity of a novel canine cathelicidin (K9CATH). The full-length cDNA sequence of K9CATH encodes a predicted 172 amino acid pre-propeptide that is 60–70% similar to other mammalian cathelicidins. Mass spectrometry analysis confirmed that the 38 aa mature K9CATH peptide was present in neutrophil granule contents. Synthetic K9CATH displayed broad antimicrobial activity against Gram-positive bacteria ( Listeria monocytogenes , and Staphylococcus aureus ; MICs (minimal inhibitory concentrations) 0.5 and 50 μM, respectively), Gram-negative bacteria ( Escherichia coli , Klebsiella pneumoniae , Salmonella serotype Typhimurium, Pseudomonas aeruginosa , Proteus mirabilis ; MICs 1.25 μM, Salmonella serotype Enteritidis; MIC 0.5 μM, and Neisseria gonorrhoeae ; MIC 0.06 μM), and yeast ( Candida albicans ; MIC 12.5–50 μM). K9CATH demonstrated high antimicrobial activity against Ureaplasma canigenitalium , and lower activity against Ureaplasma urealyticum (MIC 0.06 and 50 μM, respectively). Similar to its ovine congener SMAP-29, K9CATH possesses salt-independent antimicrobial activity and LPS binding capacity. K9CATH displayed minimal hemolytic activity against human, dog and chicken erythrocytes. The potency and broad antimicrobial activity of K9CATH suggest that this peptide may act as a fundamental contributor to the innate immune responses in this carnivore species

Cathelicidins are an important family of cationic host defense peptides in vertebrates with both antimicrobial and immunomodulatory activities. Fowlicidin-1 and fowlicidin-2 are two newly identified chicken cathelicidins with potent antibacterial activities. Here we report structural and functional characterization of the putatively mature form of the third chicken cathelicidin, fowlicidin-3, for exploration of its therapeutic potential. NMR spectroscopy revealed that fowlicidin-3 comprises 27 amino-acid residues and adopts a predominantly α-helical structure extending from residue 9 to 25 with a slight kink induced by a glycine at position 17. It is highly potent against a broad range of Gram-negative and Gram-positive bacteria in vitro, including antibiotic-resistant strains, with minimum inhibitory concentrations in the range 1–2 µm. It kills bacteria quickly, permeabilizing cytoplasmic membranes immediately on coming into contact with them. Unlike many other host defense peptides with antimicrobial activities that are diminished by serum or salt, fowlicidin-3 retains bacteria-killing activities in the presence of 50% serum or physiological concentrations of salt. Furthermore, it is capable of suppressing lipopolysaccharide-induced expression of proinflammatory genes in mouse macrophage RAW264.7 cells, with nearly complete blockage at 10 µm. Fowlicidin-3 appears to be an excellent candidate for future development as a novel antimicrobial and antisepsis agent, particularly against antibiotic-resistant pathogens.

In oviparous species, proteins and lipids are found in the vitellus forming lipoproteins called lipovitellins. They are an important energy source for embryos development and larvae growth and survival. We have previously isolated and partially characterized the sole egg cytosolic lipovitellin from the freshwater shrimp Macrobrachium borellii. It is a native protein of 440 kDa, composed of two subunits of 94 and 112 kDa. In the present work we studied size, shape and structure of M. borellii lipovitellin using electron microscopy, crosslinking reagents, MALDI-TOF, circular dichroism, fluorescence and partial proteolysis. The results showed that lipovitellin has a quasi spherical morphology with an estimated diameter of 18.5 ± 3.5 nm. It appears to be composed of two subunits of 94 kDa, and one of 112 kDa. The larger subunit is more susceptible to trypsinolysis, indicating that it is less compactly folded and/or more exposed to the aqueous medium than the 94 kDa subunits. The hetero-trimer is held together by non-covalent interactions. Peptide mass fingerprinting by MALDI-TOF, produced 42 polypeptides matching to a vitellogenin of a related species ( Macrobrachium rosenbergii ). Circular dichroism indicated that this protein contains 35.7% α-helix, 16.6% β-sheet and 20% turns. Tryptophan fluorescence emission, at a maximum of 334 nm, indicated that the environment polarity of these aromatic residues is similar to that of other crustacean lipoproteins.

The insect fat body and the adipose tissue of vertebrates store fatty acids (FA) as triacylglycerols (TG). However, the fat body of most insects has the unique ability to rapidly produce and secrete large amounts of diacylglycerol (DG). Monoacylglycerol acyltransferase (MGAT), which catalyzes the synthesis of DG from MG, and a diacylglycerol acyltransferase (DGAT), which catalyzes the synthesis of TG from DG, are key enzymes in the metabolism of neutral glycerides. However, very little is known about these acyltransferases in insects. In the present study we have cloned two predicted MGATs and a DGAT from Manduca sexta and compared their sequences with predicted MGAT and DGAT homologs from a number of insect species. The comparison suggested that insects may only have a single DGAT gene, DGAT1. The apparent absence of a DGAT2 gene in insects would represent a major difference with vertebrates, which contain DGAT1 and DGAT2 genes. Insects seem to have a single MGAT gene which is similar to the MGAT2 of vertebrates. A number of conserved phosphorylation sites of potential physiological significance were identified among insect proteins and among insect and vertebrate proteins. DGAT1 and MGAT are expressed in fat body, midgut and ovaries. The relative rates of utilization of FAs for the synthesis of DG and TG correlated with the relative expression levels of MGAT and DGAT suggesting that regulation of the expression levels of these acyltransferases could be determining whether the fat body secretes DG or stores fatty acids as TG. The expression patterns of the acyltransferases suggest a role of the monoacylglycerol pathway in the production and mobilization of DG in M. sexta fat body.

Triglyceride-lipase (TGL) is a major fat body lipase in Manduca sexta. The knowledge of how TGL activity is regulated is very limited. A WWE domain, presumably involved in protein-protein interactions, has been previously identified in the N-terminal region of TGL. In this study, we searched for proteins partners that interact with the N-terminal region of TGL. Thirteen proteins were identified by mass spectrometry, and the interaction with four of these proteins was confirmed by immunoblot. The oxidoreductase lipoamide-dehydrogenase (LipDH) and the apolipoprotein components of the lipid transporter, HDLp, were among these proteins. LipDH is the common component of the mitochondrial α-keto acid dehydrogenase complexes whereas HDLp occurs in the hemolymph. However, subcellular fractionation demonstrated that these two proteins are relatively abundant in the soluble fraction of fat body adipocytes. The cofactor lipoate found in typical LipDH substrates was not detected in TGL. However, TGL proved to have critical thiol groups. Additional studies with inhibitors are consistent with the notion that LipDH acting as a diaphorase could preserve the activity of TGL by controlling the redox state of thiol groups. On the other hand, when TG hydrolase activity of TGL was assayed in the presence of HDLp, the production of diacylglycerol (DG) increased. TGL-HDLp interaction could drive the intracellular transport of DG. TGL may be directly involved in the lipoprotein assembly and loading with DG, a process that occurs in the fat body and is essential for insects to mobilize fatty acids. Overall the study suggests that TGL occurs as a multi-protein complex supported by interactions through the WWE domain.

Manduca Sexta adults insects have two defined lipophorin species of densities 1.09 g/mL, [high-density lipophorin (HDLp)] and 1.02 g/mL [low-density lipophorin (LDLp)], respectively, and a continuous broad range of lipophorin particles of intermediate size and density, intermediate-density lipophorin (IDLp). The transformation of HDLp into IDLp and LDLp is the result of the progressive loading of HDLp with diacylglycerol (DG) and an exchangeable apolipoprotein, apolipophorin-III (apoLp-III). In this paper, we describe the physiochemical changes which occur in the lipophorin surface as a result of the transformation of HDLp into LDLp. (1) The increase in apoLp-III content, from 0 to 16 molecules per particle, is accompanied by a gradual increase in the zeta-potential which, at pH 8.6 ranges from /1.02 mV for lipophorins without apoLp-III to -7.76 mV for lipophorins containing 16 molecules of apoLp-III. (2) As judged by the changes in the partition constant for trimethylammonium diphenylhexatriene and oleic acid, an average 2-fold increase in the size of the lipophorin lipid surface takes place when HDLp is loaded with Dg and transformed into LDLp. (3) These data, as well as the results obtained by end point lipolysis with a triacylglycerol (TG) lipase, indicated that the accessible DG content increases 4-7 times when HDLp is converted in LDLp. (4) Fluorescence polarization of the cationic and anionic lipid probes, trimethylammonium diphenylhexatriene and cis-parinaric acid, embedded in eight different subspecies of lipophorin, containing from 12 to 50% DG, showed a small decrease in the surface lipid order when going from HDLp (25% DG) to LDLp (50% DG). (5) Porcine pancreatic phospholipase A2 was used as a probe of the lipoprotein surface. As the DG content of the lipoprotein increased, a higher enzyme activity against the lipoprotein-phospholipids was observed, with a maximum activity 5-fold higher against LDLp than against HDLp. Overall, the changes observed as the lipoprotein particles are loaded with DG and apoLp-III provide a link between the structure and properties of the lipophorin surface and the physiological roles of HDLp and LDLp particles.

The lipid droplets (LDs) are intracellular organelles mainly dedicated to the storage and provision of fatty acids. To accomplish these functions the LDs interact with other organelles and cytosolic proteins. In order to explore possible correlations between the physiological states of cells and the protein composition of LDs we have determined and compared the proteomic profiles of lipid droplets isolated from the fat bodies of 5th-instar larvae and adult Manduca sexta insects and from ovaries. These LD-rich tissues represent three clearly distinct metabolic states in regard to lipid metabolism: 1) Larval fat body synthesizes fatty acids (FA) and accumulates large amounts as triglyceride (TG); 2) Fat body from adult insects provides FA to support reproduction and flight; 3) Ovaries do not synthesize FA, but accumulate considerable amounts of TG in LDs. Major qualitative and semi-quantitative variations in the protein compositions of the LDs isolated from these three tissues were observed by MS/MS and partially validated by immuno-blotting. The differences observed included changes in the abundance of lipid droplet specific proteins, cytosolic proteins, mitochondrial proteins and also proteins associated with the machinery of protein synthesis. These results suggest that changes in the interaction of LDs with other organelles and cytosolic proteins are tightly related to the physiological state of cells. Herein, we summarize and compare the protein compositions of three subtypes of LDs and also describe for the first time the proteomic profile of LDs from an insect ovary. The compositions and compositional differences found among the LDs are discussed to provide a platform for future studies on the role of LDs, and their associated proteins, in cellular metabolism.

Background In 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. Results We 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. Conclusions We 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.

Adipokinetic hormone (AKH) is the main hormone involved in the acute regulation of hemolymph lipid levels in several insects. In adult Manduca sexta AKH promotes a rapid phosphorylation of “Lipid storage protein-1”, Lsd1, and a concomitant activation of the rate of hydrolysis of triglycerides by the main fat body lipase. In contrast, in the larval stage AKH modulates hemolymph trehalose levels. The present study describes the sequence of a full-length Lsd1 cDNA obtained from M. sexta fat body and investigates a possible link between Lsd1 expression and the distinct effects of AKH in larva and adult insects. The deduced protein sequence showed a high degree of conservation compared to other insect Lsd1s, particularly in the central region of the protein (amino acids 211–276) in which the predicted lipid binding helices are found. Lsd1 was absent in feeding larva and its abundance progressively increased as the insect develops from the non-feeding larva to adult. Contrasting with the levels of protein, Lsd1 transcripts were maximal during the feeding larval stages. The subcellular distribution of Lsd1 showed that the protein exclusively localizes in the lipid droplets. Lsd1 was found in the fat body but it was undetectable in lipid droplets isolated from oocytes or embryos. The present study suggests a link between AKH-stimulated lipolysis in the fat body and the expression of Lsd1.

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 (11.1 ± 2.1 nmol TG/min-mg) was 3.1-fold higher than that determined with control cytosol and lipid droplets (3.6 ± 0.5 nmol TG/min-mg). 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. Arch. Insect Biochem. Physiol. 63:73–81, 2006. © 2006 Wiley-Liss, Inc.

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.

An original method for the study of the lipid binding properties of exchangeable apolipoproteins is reported. Binding of Locusta migratoria apolipophorin-III to Manduca sexta low-density lipophorin (LDLp) and high-density lipophorin (HDLp) was studied in vivo. This assay could be used useful to investigate the effect of mutations in the lipid binding properties of exchangeable apolipoproteins under physiological conditions.

In order to probe the organization of diacylglycerol (DG) in lipophorin, 13C-enriched lipophorin was prepared for NMR investigations. We obtained 13C-enriched lipophorin labeled exclusively in DG by feeding insects tobacco leaves coated with [1-13C]palmitic acid or [1-13C]oleic acid. Lipophorins enriched up to 5% with a [13C]fatty acid were obtained by this procedure. NMR studies of the isolated lipophorin DG showed that palmitic acid accumulates almost entirely (> 90%) in the sn-1 position. Oleic acid was found equally distributed between the sn-1 and sn-2 positions, yielding a DG enriched equally at both positions. The 13C-NMR spectra of both [13C]palmitate- and [13C]oleate-enriched lipophorins showed that DG had one narrow carbonyl resonance indicative of rapid motion. A comparative analysis of the 13C carbonyl chemical shift data for DG in organic solvents, aqueous solutions, and dispersions with the DG carbonyl chemical shift of native lipophorin enriched in [13C]palmitate or [13C]oleate shows a high degree of water exclusion from the DG carbonyls in lipophorin. This result is consistent with the existence of a lipophorin lipid core containing most of the lipophorin DG. This study represents the first attempt to elucidate the organization of DG in lipophorin. The possibility of obtaining [13C]DG-enriched lipophorins, selectively enriched in one or both acyl chains of DG, should provide a powerful tool for further analysis of the organization and the dynamic properties of DG in native lipoproteins.

The very high density lipoprotein (VHDL) of Triatoma injesfum hemolymph from adult males has been isolated and purified by two-step density gradient ultracentrifugation. It appears to be homogeneous as judged by native polyacrylamide gel electrophoresis. The content of VHDL in hemolymph was estimated to be 8 mg proteidml. The purified protein has a molecular weight (M,) of 450,000, is composed of six subunits of M, p 77,000, and possesses a high content of aromatic amino acids. This protein is glycosylated and contains 3% of lipids by weight with a remarkable amount of free fatty acids (25% of total lipids). The I: injesfans VHDL has a different lipid and amino acid composition from lipophorin. The lipid composition and the spectroscopic studies using cis-parinaric acid indicated a high fatty acid binding affinity. It has nine binding sites per mol of VHDL. Competence studies revealed that VHDL has its highest affinity for the binding of palmitic acid followed by stearic and arachidonic acids., Instituto de Investigaciones Bioquímicas de La Plata

Triatoma infestans hemolymph has 0.31 mg/ml of free fatty acids and 2.8 mg/ml of diacylglycerols. Almost all the diacylglycerols are transported by lipophorin whereas free fatty acids are carried by lipophorin and a very high density lipoprotein. The binding of cis-parinaric acid to lipophorin was employed to specify the free fatty acid binding properties of lipophorin. Lipophorin has 10 binding sites of high affinity (3 x 10(7)) and approximately 45 binding sites of low affinity (1 x 10(6)). The relative rate of tissue incorporation of free fatty acids and diacylglycerols was measured by injecting insects with hemolymph previously labeled in both, free fatty acids and diacylglycerols. In this way, the half-life of the hemolymph free fatty acids was estimated to be about 4 min. Based on this result and taking into account the content of free fatty acids and diacylglycerols in hemolymph, the incorporation of free fatty acids, expressed in moles of fatty acids, seems to be 3.4 times higher than that of diacylglycerols. This finding can be applied to other insects.