Kozo Tsuchida

Lipid storage and mobilization in insects: current status and future directions

In this paper we review the current status of research on fatty acid absorption and conversion to diacylglycerol in the midgut. We further discuss how diacylglycerol may leave the midgut and associate with lipophorin in hemolymph. We review the present understanding of the role of the lipid transfer particle and lipophorin receptors in lipid delivery between lipophorin and tissues. Finally, we discuss recent studies on the mobilization of diacylglycerol from the fat body in response to adipokinetic hormone. Several suggestions for exciting areas of future research are described.

Digestion, absorption, transport and storage of fat during the last larval stadium of Manduca sexta. Changes in the role of lipophorin in the delivery of dietary lipid to the fat body

Abstract Using radiolabeled triolein, it was shown that in Manduca sexta larvae dietary triacylglycerol was hydrolyzed completely to free fatty acid in the midgut before absorption into midgut cells. Subsequently, the fatty acids enter the midgut cell where they are found in phospholipid, di- and triacylglycerol and leave the midgut as diacylglycerol and are transported by lipophorin. In feeding larvae, lipophorin diacylglycerol was rapidly cleared from the hemolymph and most of the label was found in fat body triacylglycerol. In contrast, diacylglycerol was cleared very slowly from wanderer hemolymph and only a very small amount was taken up by the fat body. In vitro studies showed that these differences in uptake of diacylglycerol from lipophorin were a result of alterations in properties of fat body during development. Further in vitro studies showed that when lipophorin was incubated with feeding stage fat body more than 95% of the diacylglycerol was taken up by the fat body but none of the lipophorin protein entered the fat body.

A carotenoid-binding protein (CBP) plays a crucial role in cocoon pigmentation of silkworm (Bombyx mori) larvae

We examined the role of carotenoid‐binding protein (CBP) in yellow cocoon pigmentation. First, using yellow or white cocoon races, we investigated the linkage between the yellow pigmentation and CBP expression. CBP was expressed only in the silk gland of the yellow cocoon races, which utilize carotenoids for cocoon pigmentation. Furthermore, CBP expression in the silk glands of day 1–7 fifth instar larvae matched the period of carotenoid uptake into the silk gland. Finally, we gave double‐stranded CBP RNA to Bombyx mori (B. mori) larvae to induce RNA interference. The significantly reduced expression of CBP in the silk gland of fifth instar larva was confirmed on day 4 and a decrease in yellow pigmentation was observed in the cocoon. We showed that CBP plays a key role in the yellow cocoon pigmentation caused by carotenoids.

Purification and partial characterization of a lutein-binding protein from human retina

Dietary intake of lutein and zeaxanthin appears to be advantageous for protecting human retinal and macular tissues from degenerative disorders such as age-related macular degeneration. Selective concentration of just two of the many dietary carotenoids suggests that uptake and transport of these xanthophyll carotenoids into the human foveal region are mediated by specific xanthophyll-binding proteins such as GSTP1 which has previously been identified as the zeaxanthin-binding protein of the primate macula. Here, a membrane-associated human retinal lutein-binding protein (HR-LBP) was purified from human peripheral retina using ion-exchange chromatography followed by size-exclusion chromatography. After attaining 83-fold enrichment of HR-LBP, this protein exhibited a significant bathochromic shift of approximately 90 nm in association with lutein, and equilibrium binding studies demonstrated saturable, specific binding toward lutein with a K(D) of 0.45 muM. Examination for cross-reactivity with antibodies raised against known lutein-binding proteins from other organisms revealed consistent labeling of a major protein band of purified HR-LBP at approximately 29 kDa with an antibody raised against silkworm (Bombyx mori) carotenoid-binding protein (CBP), a member of steroidogenic acute regulatory (StAR) protein family with significant homology to many human StAR proteins. Immunolocalization with antibodies directed against either CBP or GSTP1 showed specific labeling of rod and cone inner segments, especially in the mitochondria-rich ellipsoid region. There was also strong labeling of the outer plexiform (Henle fiber) layer with anti-GSTP1. Such localizations compare favorably with the distribution of macular carotenoids as revealed by resonance Raman microscopy. Our results suggest that HR-LBP may facilitate luteins localization to a region of the cell subject to considerable oxidative stress.

Effect of dietary lipid content on lipid transport and storage during larval development of Manduca sexta

Manduca sexta larvae were raised on diets containing either 1.2% fat (control diet), 5.9% fat (high-fat diet) or on a fat-free diet. Insects raised on the control and high-fat diets did not differ significantly in body weight, whereas animals raised on the fat-free diet were significantly smaller. The fat content of the diet had no effect on the hemolymph concentration of lipophorin. During the larval period, lipophorin isolated from animals on the high-fat diet contained more lipid, and lipophorin isolated from animals on the fat-free diet contained less lipid than lipophorin isolated from control animals. However, lipophorin isolated from animals during the prepupal period had the same composition regardless of diet. Compared to controls, animals on the high-fat diet had a larger mass of fat body which contained more stored triacylglycerol, while animals on the fat-free diet had a smaller mass of fat body which contained less stored triacylglycerol. As the fat content of the diet was increased, the fatty acid composition of fat body triacylglycerols reflected more closely that of the dietary lipid.

A CD36-related Transmembrane Protein Is Coordinated with an Intracellular Lipid-binding Protein in Selective Carotenoid Transport for Cocoon Coloration

The transport pathway of specific dietary carotenoids from the midgut lumen to the silk gland in the silkworm, Bombyx mori, is a model system for selective carotenoid transport because several genetic mutants with defects in parts of this pathway have been identified that manifest altered cocoon pigmentation. In the wild-type silkworm, which has both genes, Yellow blood (Y) and Yellow cocoon (C), lutein is transferred selectively from the hemolymph lipoprotein to the silk gland cells where it is accumulated into the cocoon. The Y gene encodes an intracellular carotenoid-binding protein (CBP) containing a lipid-binding domain known as the steroidogenic acute regulatory protein-related lipid transfer domain. Positional cloning and transgenic rescue experiments revealed that the C gene encodes Cameo2, a transmembrane protein gene belonging to the CD36 family genes, some of which, such as the mammalian SR-BI and the fruit fly ninaD, are reported as lipoprotein receptors or implicated in carotenoid transport for visual system. In C mutant larvae, Cameo2 expression was strongly repressed in the silk gland in a specific manner, resulting in colorless silk glands and white cocoons. The developmental profile of Cameo2 expression, CBP expression, and lutein pigmentation in the silk gland of the yellow cocoon strain were correlated. We hypothesize that selective delivery of lutein to specific tissue requires the combination of two components: 1) CBP as a carotenoid transporter in cytosol and 2) Cameo2 as a transmembrane receptor on the surface of the cells.

Lipid transfer particle catalyzes transfer of carotenoids between lipophorins of Bombyx mori.

The yellow color of Bombyx mori hemolymph is due to the presence of carotenoids, which are primarily associated with lipophorin particles. Carotenoids were extracted from high density lipophorin (HDLp) of B. mori and analyzed by HPLC. HDLp contained 33 micrograms of carotenoids per mg protein. Over 90% of carotenoids were lutein while alpha-carotene and beta-carotene were minor components. When larval hemolymph was subjected to density gradient ultracentrifugation, a second minor yellow band was present, which was identified as B. mori lipid transfer particle (LTP). During other life stages examined however, this second band was not visible. To determine if coloration of LTP may fluctuate during development, we determined its concentration in hemolymph and compared it to that of lipophorin. Both proteins were present during all life stages and their concentrations gradually increased. The ratio of lipophorin: LTP was 10-15:1 during the fourth and fifth instar larval stages, and 20-30:1 during the pupal and adult stages. Thus, there was no correlation between the yellow color attributed to LTP and its hemolymph concentration. It is possible that yellow coloration of the LTP fraction corresponds to developmental stages when the particle is active in carotene transport. To determine if LTP is capable of facilitating carotene transfer, we took advantage of a white hemolymph B. mori strain which, when fed artificial diet containing a low carotene content, gives rise to a lipophorin that is nearly colorless. A spectrophotometric, carotene specific, transfer assay was developed which employed wild type, carotene-rich HDLp as donor particle and colorless low density lipophorin, derived from the white hemolymph strain animals, as acceptor particle. In incubations lacking LTP carotenes remained associated with HDLp while inclusion of LTP induced a redistribution of carotenes between the donor and acceptor in a time and concentration dependent manner. Time course studies suggested the rate of LTP-mediated carotene transfer was relatively slow, requiring up to 4 h to reach equilibrium. By contrast, studies employing 3H-diacylglycerol labeled HDLp as donor particle in lipid transfer assays revealed a rapid equilibration of label between the particles. Thus, it is plausible that the slower rate of LTP-mediated carotene transfer is due to its probable sequestration in the core of HDLp.

Lipophorin receptor of Bombyx mori: cDNA cloning, genomic structure, alternative splicing, and isolation of a new isoform.

The cDNA and genomic structure of a putative lipophorin receptor from the silkworm, Bombyx mori (BmLpR), indicated the presence of four isoforms, designated LpR1, LpR2, LpR3, and LpR4. The deduced amino acid sequence of each isoform showed five functional domains that are homologous to vertebrate very low density lipoprotein receptor (VLDLR). All four isoforms seem to have originated from a single gene by alternative splicing and were differentially expressed in a tissue- and stage-specific manner. BmLpR1 harbored an additional 27 amino acids in the O-linked sugar domain, resulting in an extra exon. The silkworm BmLpR gene consisted of 16 exons separated by 15 introns spanning >122 kb and was at least three times larger than the human VLDLR gene. Surprisingly, one of the isoforms, LpR4, was expressed specifically in the brain and central nervous system. Additionally, it had a unique cytoplasmic tail, leading to the proposition that it represents a new candidate LpR for possible brain-related function(s). This is the first report on the genomic characterization of an arthropod lipoprotein receptor gene and the identification of a brain-specific receptor variant from a core member of the low density lipoprotein receptor family in invertebrates.

A defective non-LTR retrotransposon is dispersed throughout the genome of the silkworm, Bombyx mori

The presence of long repetitive sequences is demonstrated in the genome of the silkworm, Bombyx mori. Members of this BMC1 family reveal several features typical of the L1 (long interspersed sequence one) family of mammals, except for species specific elements. The number of BMC1 elements is estimated to be approximately 3500 per haploid genome. Elements containing the full length unit of 5.1 kb are dispersed throughout the genome and their restriction sites are conserved, although most members are preferentially truncated to varying extents at their 5′ ends. DNA sequencing indicates that this element contains six tandem repeats of 15 bp CpG-rich sequence in the 5′ proximal region. It terminates with a 3′ oligo(A) stretch, and is flanked at both ends by a 7–10 bp target sequence duplication. In addition, there is significant evidence for amino acid sequence homology with reverse transcriptase domains of other L1 families, especially F, Doc and lockey of Drosophila melanogaster. No large open reading frame is present. The BMC1 element is suggested to be dispersed in the genome by a transposition mechanism involving RNA intermediates.

Transfer of cholesterol and diacylglycerol from lipophorin to Bombyx mori ovarioles in vitro: role of the lipid transfer particle

The objective of this study was to characterize the transfer of diacylglycerol (DAG) and cholesterol from larval Bombyx mori lipophorin to ovarioles. Transfer studies were carried out by incubating pupal ovarioles (5-day) with [(3)H]-cholesterol and [(3)H]-DAG-labeled lipophorin under different conditions. Transfer of both cholesterol and DAG exhibited hyperbolic dependency on lipophorin concentration with apparent Km values of 0.83 +/- 0.17 mg/ml and 0.74 +/- 0.16 mg/ml, respectively. Pretreatment of ovarioles with anti-lipid transfer particle (LTP) IgG significantly inhibited transfer of labeled DAG to ovarioles (75%) and not cholesterol. Injection of B. mori pupae (day 4) with anti-LTP IgG significantly affected the weight (65%), number of eggs (49%), amount of lipid (74%), and protein (65%) of the adult ovaries. Matured eggs had a very faint yellow color and deformed shape compared to controls. The inhibitory effect demonstrates the active role LTP plays in growth of ovaries, development, and oogenesis. The effect on vitellogenin shortage on egg development and maturation was determined by implanting ovaries in male recipients that lack vitellogenin. An 80% decline in egg production was observed. However, the mature eggs were normal in shape, color, and lipid content. Thus, restricting lipid or protein delivery to developing ovaries would dramatically affect choriogenesis.