Lipid transfer particle mediates the delivery of diacylglycerol from lipophorin to fat body in larval Manduca sexta
2004; Elsevier BV; Volume: 45; Issue: 3 Linguagem: Inglês
10.1194/jlr.m300242-jlr200
ISSN1539-7262
AutoresLilián E. Canavoso, Hwa Kyung Yun, Zeina E. Jouni, Michael A. Wells,
Tópico(s)Lipid Membrane Structure and Behavior
ResumoThis work analyzed the process of lipid storage in fat body of larval Manduca sexta, focusing on the role of lipid transfer particle (LTP). Incubation of fat bodies with [3H]diacylglycerol-labeled lipophorin resulted in a significant accumulation of diacylglycerol (DAG) and triacylglycerol (TAG) in the tissue. Transfer of DAG to fat body and its storage as TAG was significantly inhibited (60%) by preincubating the tissue with anti-LTP antibody. Lipid transfer was restored to control values by adding LTP to fat body. Incubation of fat body with dual-labeled DAG lipophorin or its treatment with ammonium chloride showed that neither a membrane-bound lipoprotein lipase nor lipophorin endocytosis is a relevant pathway to transfer or to storage lipids into fat body, respectively. Treatment of fat body with suramin caused a 50% inhibition in [3H]DAG transfer from lipophorin. Treatment of [3H]DAG-labeled fat body with lipase significantly reduced the amount of [3H]DAG associated with the tissue, suggesting that the lipid is still on the external surface of the membrane.Whether this lipid represents irreversibly adsorbed lipophorin or a DAG lipase-sensitive pool is unknown. Nevertheless, these results indicate that the main pathway for DAG transfer from lipophorin to fat body is via LTP and receptor-mediated processes. This work analyzed the process of lipid storage in fat body of larval Manduca sexta, focusing on the role of lipid transfer particle (LTP). Incubation of fat bodies with [3H]diacylglycerol-labeled lipophorin resulted in a significant accumulation of diacylglycerol (DAG) and triacylglycerol (TAG) in the tissue. Transfer of DAG to fat body and its storage as TAG was significantly inhibited (60%) by preincubating the tissue with anti-LTP antibody. Lipid transfer was restored to control values by adding LTP to fat body. Incubation of fat body with dual-labeled DAG lipophorin or its treatment with ammonium chloride showed that neither a membrane-bound lipoprotein lipase nor lipophorin endocytosis is a relevant pathway to transfer or to storage lipids into fat body, respectively. Treatment of fat body with suramin caused a 50% inhibition in [3H]DAG transfer from lipophorin. Treatment of [3H]DAG-labeled fat body with lipase significantly reduced the amount of [3H]DAG associated with the tissue, suggesting that the lipid is still on the external surface of the membrane. Whether this lipid represents irreversibly adsorbed lipophorin or a DAG lipase-sensitive pool is unknown. Nevertheless, these results indicate that the main pathway for DAG transfer from lipophorin to fat body is via LTP and receptor-mediated processes. In insects, the majority of stored lipids are found in the fat body, an organ analogous to vertebrate adipose tissue and liver. In Manduca sexta, triacylglycerol (TAG) constitutes more than 90% of the fat body lipids, whereas diacylglycerol (DAG) accounts for less than 2–3%. TAG is derived mainly from dietary fat, which is transferred in the form of DAG by lipophorin from the midgut to the fat body during the feeding stage. A minor source of TAG in the fat body is the de novo lipid synthesis from carbohydrates (1Beenakkers A.M.T. Van der Horst D.J. Van Marrewijk W.J.A. Insect lipids and their role in physiological processes.Prog. Lipid Res. 1985; 24: 19-67Crossref PubMed Scopus (317) Google Scholar). We have shown recently that the uptake of DAG from midgut to lipophorin takes place by the action of lipid transfer particle (LTP) (2Canavoso L.E. Wells M.A. Metabolic pathways for diacylglycerol biosynthesis and release in the midgut of larval Manduca sexta.Insect Biochem. Mol. Biol. 2000; 30: 1173-1180Crossref PubMed Scopus (30) Google Scholar). LTP is a very high density lipoprotein, first purified from the hemolymph of larval M. sexta (3Ryan R.O. Wells M.A. Law J.H. Lipoprotein interconversions in an insect, Manduca sexta. Evidence for a lipid transfer factor in the hemolymph.J. Biol. Chem. 1986; 261: 563-568Abstract Full Text PDF PubMed Google Scholar) [see also refs. (4Arrese E.L. Canavoso L.E. Jouni Z.E. Pennington J.E. Tsuchida K. Wells M.A. Lipid storage and mobilization in insects: current status and future directions.Insect Biochem. Mol. Biol. 2000; 31: 7-17Crossref Scopus (197) Google Scholar, 5Canavoso L.E. Karnas J.K. Jouni Z.E. Pennington J.E. Wells M.A. Fat metabolism in insects.Annu. Rev. Nutr. 2001; 21: 23-46Crossref PubMed Scopus (464) Google Scholar) for recent reviews]. LTP has also been identified in the hemolymph of Locusta migratoria (6Hirayama Y. Chino H. Lipid transfer particle in locust hemolymph: purification and characterization.J. Lipid Res. 1990; 31: 793-799Abstract Full Text PDF PubMed Google Scholar), Periplaneta americana (7Takeuchi N. Chino H. Lipid transfer particle in the hemolymph of the American cockroach: evidence for its capacity to transfer hydrocarbons between lipophorin particles.J. Lipid Res. 1993; 34: 543-551Abstract Full Text PDF PubMed Google Scholar), Musca domestica (8Capurro M. de L. De Bianchi A.G. A lipid transfer particle in Musca domestica haemolymph.Comp. Biochem. Physiol. 1990; 97B: 649-653Google Scholar), and Bombyx mori (9Tsuchida K. Soulages J.L. Moribayashi A. Suzuki K. Maekawa H. Wells M.A. Purification and properties of a lipid transfer particle from Bombyx mori: comparison to the lipid transfer particle from Manduca sexta.Biochim. Biophys. Acta. 1997; 1337: 57-65Crossref PubMed Scopus (32) Google Scholar). LTP is synthesized in the fat body and secreted into the hemolymph (10Van Heusden M.C. Yepiz-Plascencia G.M. Walker A.M. Law J.H. Manduca sexta lipid transfer particle: synthesis by fat body and occurrence in the hemolymph.Arch. Insect Biochem. Physiol. 1996; 31: 273-287Crossref PubMed Scopus (14) Google Scholar). The physiological function of LTP is still not completely understood. The protein catalyzes the transfer of DAG from adult fat body to high density lipophorin (Lp), resulting in the formation of low density lipophorin (LDLp) (11Van Heusden C.M. Law J.H. An insect lipid transfer particle promotes lipid loading from fat body to lipoprotein.J. Biol. Chem. 1989; 264: 17287-17292Abstract Full Text PDF PubMed Google Scholar), but not the reverse reaction. LTP catalyzes the transfer of DAG from the larval midgut to Lp, but not the reverse reaction (12Canavoso L.E. Wells M.A. Role of lipid transport particle in lipid delivery of diacylglycerol from midgut to lipophorin in larval Manduca sexta.Insect Biochem. Mol. Biol. 2001; 31: 783-790Crossref PubMed Scopus (39) Google Scholar), and from Lp to ovarioles (13Jouni Z.E. Takada N. Gazard J. Maekawa H. Wells M.A. Tsuchida K. Transfer of cholesterol and diacylglycerol from lipophorin to Bombyx mori oocytes in vitro: role of the lipid transfer particle.Insect Biochem. Mol. Biol. 2003; 33: 145-153Crossref PubMed Scopus (28) Google Scholar). LTP also catalyzes the transfer and/or exchange of DAG between Lp or LDLp and vitellogenin (9Tsuchida K. Soulages J.L. Moribayashi A. Suzuki K. Maekawa H. Wells M.A. Purification and properties of a lipid transfer particle from Bombyx mori: comparison to the lipid transfer particle from Manduca sexta.Biochim. Biophys. Acta. 1997; 1337: 57-65Crossref PubMed Scopus (32) Google Scholar). The protein also facilitates the transfer of other lipids from Lp to LDLp, including hydrocarbons (7Takeuchi N. Chino H. Lipid transfer particle in the hemolymph of the American cockroach: evidence for its capacity to transfer hydrocarbons between lipophorin particles.J. Lipid Res. 1993; 34: 543-551Abstract Full Text PDF PubMed Google Scholar), phospholipids (9Tsuchida K. Soulages J.L. Moribayashi A. Suzuki K. Maekawa H. Wells M.A. Purification and properties of a lipid transfer particle from Bombyx mori: comparison to the lipid transfer particle from Manduca sexta.Biochim. Biophys. Acta. 1997; 1337: 57-65Crossref PubMed Scopus (32) Google Scholar), and carotenoids (14Tsuchida K. Arai M. Tanaka Y. Ishihara R. Ryan R.O. Maekawa H. Lipid transfer particle catalyzes transfer of carotenoids between lipophorins of Bombyx mori.Insect Biochem. Mol. Biol. 1998; 28: 927-934Crossref PubMed Scopus (48) Google Scholar), and it catalyzes the exchange and/or transfer of DAG between Lps and human lipoproteins (15Blacklock B.J. Ryan R.O. Hemolymph lipid transport.Insect Biochem. Mol. Biol. 1994; 24: 855-873Crossref PubMed Scopus (131) Google Scholar). We have been investigating the role of LTP in DAG transfer in larval M. sexta using in vitro assays. In the midgut, LTP is required to export DAG from the midgut to Lp (12Canavoso L.E. Wells M.A. Role of lipid transport particle in lipid delivery of diacylglycerol from midgut to lipophorin in larval Manduca sexta.Insect Biochem. Mol. Biol. 2001; 31: 783-790Crossref PubMed Scopus (39) Google Scholar). Interestingly, LTP does not catalyze the transfer of DAG from Lp to the midgut. In this paper, we describe the extension of these studies to the larval fat body and show for the first time that LTP is required for the bidirectional transfer of DAG between Lp and fat body. [9,10-3H]Oleic acid was purchased from NEN (Boston, MA). [1-14C]Oleic acid and [1(3)-3H]glycerol were from Amersham (Arlington Heights, IL). Silica gel plates were obtained from J. T. Baker. 4-(2-Aminoethyl)-benzenesulfonylfluoride (AEBSF) and DEAE-Trisacryl M were from Sigma (St. Louis, MO). Falcon multiwell tissue culture plates and cell strainers were obtained from Becton Dickinson (Franklin Lakes, NJ). Affi-Gel Protein A was from Bio-Rad (Hercules, CA). Centriprep Centrifugal Filter Devices were from Millipore-Amicon (Bedford, MA). Rhizopus lipase was purchased from Fluka (Buchs, Switzerland). All other chemicals were analytical grade. M. sexta was reared as previously described (16Prasad S.V. Ryan R.O. Law J.H. Wells M.A. Changes in lipophorin composition during larval-pupal metamorphosis of an insect, Manduca sexta.J. Biol. Chem. 1986; 261: 558-562Abstract Full Text PDF PubMed Google Scholar). For the experiments, day 1 or day 2 fifth instar larvae were used unless otherwise indicated. Two-day-old fifth instar larvae were fed [3H]oleic acid (5 μCi/animal) on a small piece of artificial diet. One hour later, hemolymph was collected in ice-cold bleeding solution (30 mM KH2PO4, pH 6.5, containing 2 mM Na2EDTA, 10 mM glutathione, and 3 mM NaN3) by puncturing a proleg and gently pressing the abdomen. Hemolymph was centrifuged for 5 min at 12,000 g (4°C) to remove hemocytes and then subjected to two steps of KBr gradient ultracentrifugation as described previously (16Prasad S.V. Ryan R.O. Law J.H. Wells M.A. Changes in lipophorin composition during larval-pupal metamorphosis of an insect, Manduca sexta.J. Biol. Chem. 1986; 261: 558-562Abstract Full Text PDF PubMed Google Scholar, 17Shapiro J.P. Keim P.S. Law J.H. Structural studies on lipophorin, an insect lipoprotein.J. Biol. Chem. 1984; 259: 3680-3685Abstract Full Text PDF PubMed Google Scholar). Labeled Lp (density of 1.14 g/ml) was dialyzed against lepidopteran saline (5 mM KH2PO4, 100 mM KCl, 4 mM NaCl, 15 mM MgCl2, and 2 mM CaCl2, pH 6.5) and concentrated by ultrafiltration before use (Centriprep YM-50). Under these conditions, more than 95% of the label was recovered in the DAG-Lp moiety, and this material is designated [3H]DAG-Lp. LTP was isolated from hemolymph of 2-day-old fifth instar M. sexta larvae (12Canavoso L.E. Wells M.A. Role of lipid transport particle in lipid delivery of diacylglycerol from midgut to lipophorin in larval Manduca sexta.Insect Biochem. Mol. Biol. 2001; 31: 783-790Crossref PubMed Scopus (39) Google Scholar, 18Tsuchida K. Wells M.A. Digestion, absorption, transport and storage of fat during the last larval stadium of Manduca sexta. Changes in the role of lipophorin in the delivering of dietary lipid to the fat body.Insect Biochem. 1988; 18: 263-268Crossref Scopus (77) Google Scholar). Briefly, 20 ml of hemocyte-free hemolymph was adjusted to a density of 1.31 g/ml with KBr, transferred to a 39 ml Quick-Seal centrifuge tube, and overlaid with 0.15 M NaCl. Centrifugation was carried out for 4 h at 50,000 rpm (4°C) in a Beckman VTi 50 rotor. The LTP fraction (density of 1.23 g/ml) was collected and adjusted to 1.31 g/ml with KBr, and 20 ml fractions of this preparation were overlaid with a 1.21 g/ml KBr solution in 0.15 M NaCl. A second ultracentrifugation was carried out for 16 h, and LTP was recovered from the top 5 ml of the gradient and dialyzed against 20 mM Tris-HCl, pH 8.7, containing 5 mM Na2EDTA. LTP was purified before each experiment by a double passage through a DEAE-Trisacryl M column (14Tsuchida K. Arai M. Tanaka Y. Ishihara R. Ryan R.O. Maekawa H. Lipid transfer particle catalyzes transfer of carotenoids between lipophorins of Bombyx mori.Insect Biochem. Mol. Biol. 1998; 28: 927-934Crossref PubMed Scopus (48) Google Scholar). The protein was eluted with a linear 0–300 mM NaCl gradient (flow rate of 15 ml/h), and 3 ml fractions were collected. LTP-containing fractions were pooled and stored at 4°C in 3.7 M KBr and 1 mM AEBSF for no longer than 1 week. Before its use, LTP was dialyzed against lepidopteran saline and concentrated by ultrafiltration (Centriprep YM-50). Fat bodies from second day fifth instar larvae were dissected on ice-cold lepidopteran saline and then transferred to a 24-well culture plate containing the incubation medium (lepidopteran saline-Grace's insect medium in a 1:1 ratio). After 5 min, each fat body was transferred to 1.5 ml of fresh medium containing [3H]DAG-Lp (1.5 mg/ml; specific activity of 5.5 × 105 dpm/mg Lp). Incubations were performed at room temperature or 4°C (control) with gentle shaking and continuous oxygenation by aeration with 95% O2-5% CO2 (19Canavoso L.E. Rubiolo E.R. Interconversions of lipophorin particles by adipokinetic hormone in hemolymph of Panstrongylus megistus, Dipetalogaster maximus and Triatoma infestans (Hemiptera:Reduviidae).Comp. Biochem. Physiol. 1995; 112A: 143-150Crossref Scopus (21) Google Scholar). At different times, fat bodies were removed and washed twice for 15 min each with 1.5 ml of incubation medium. Preliminary data have shown that no significant further reduction in radioactivity was observed when the tissues were subjected to more than two washes. Thus, two washing steps with incubation medium was adopted in these experiments. Fat bodies were individually homogenized for lipid extraction according to Folch, Lees, and Sloane Stanley (20Folch J. Lees M. Sloane Stanley G.H. A simple method for isolation and purification of total lipids from animal tissues.J. Biol. Chem. 1957; 226: 497-509Abstract Full Text PDF PubMed Google Scholar). Lipids were fractionated by TLC, and spots scraped from the plates were assayed for radioactivity by liquid scintillation counting (2Canavoso L.E. Wells M.A. Metabolic pathways for diacylglycerol biosynthesis and release in the midgut of larval Manduca sexta.Insect Biochem. Mol. Biol. 2000; 30: 1173-1180Crossref PubMed Scopus (30) Google Scholar). Cellular viability and tissue integrity were determined by trypan blue exclusion tests. Dissected fat bodies were transferred to 1.5 ml of fresh medium and incubated as described above under the following conditions: a) for 3 h in the presence of [3H]DAG-Lp (1.5 mg/ml); b) for 1 h with anti-LTP antibody (3.5 μg IgG/μl), washed with Grace's medium, and then incubated with [3H]DAG-Lp for 3 h; and c) preincubated with anti-LTP antibody (1 h), washed, and then transferred to a medium with both [3H]DAG-Lp (1.5 mg/ml) and LTP (70 μg/ml). After incubation, fat bodies were removed, washed as described above, and processed for lipid analysis. Insects were fed a mixture of [1-14C]oleic acid (3 μCi/insect) and [1(3)-3H]glycerol (12 μCi/insect) on a small piece of artificial diet 1 h before bleeding. Isolation of doubly labeled DAG-Lp ([14C]/[3H]DAG-Lp) that incorporated the 3H label in glycerol backbone and the 14C label in the fatty acids was performed as described above. Dissected fat bodies were incubated with 1.5 ml of incubation medium containing [14C]/[3H]DAG-Lp (1 mg/ml; 14C/3H ratio of 0.97) with or without unlabeled glycerol (4 μmol/ml). After 1.5 and 3 h of incubation, tissue was removed and washed with unlabeled medium, and lipids were extracted and isolated by TLC. The radioactivity was recorded as 14C/3H ratio in DAG-Lp and fat body DAG and TAG. To determine how much residual [3H]DAG remained bound to the fat body after extensive washing, labeled fat bodies were incubated with Rhizopus lipase, which is able to hydrolyze only the surface-bound lipids. Briefly, TAG and DAG pools of the fat body were radiolabeled by incubating the tissues with [3H]DAG-Lp (1.5 mg/ml) for 2 h, followed by extensive washing with Grace's medium. The washed fat body was transferred to a tube containing 200 mg of fatty acid-free BSA in the absence (control) or presence of Rhizopus lipase (1.2 U) and incubated at 34°C for 30 min. After incubation, the reaction was stopped by adding diisopropyl fluorophosphates (5 mM), PMSF (5 mM), diethyl p-nitrophenyl phosphate (Paraoxon; E600) (3 mM), and EDTA (25 mM). Tissues were then removed, washed with Grace's medium, and processed for lipid analysis as described above. In another set of experiments, the fat body was incubated with anti-LTP (4 mg IgG/ml) for 30 min before incubation with [3H]DAG-Lp (1.5 mg/ml) and then with lipase. To study the effect of pH on the transfer of lipids from Lp to fat body, [3H]DAG-Lp was dialyzed into a buffer containing 10 mM MES, 10 mM MOPS, 0.5 mM Trizma base, and 0.15 M NaCl of different pH values between 5.0 and 7.5. Transfer studies were carried out as described above. To study the effect of inhibitors of endocytosis, the fat body was incubated for 2 h in insect Grace's medium containing [3H]DAG-Lp (1.5 mg/ml) containing different concentrations of ammonium chloride or chloroquine. After incubation, the fat body was washed and lipids were analyzed as described above. To study the extent of transfer of [3H]DAG from different developmental stages of fat body to Lp, day 1 fifth instar M. sexta were fed on a piece of diet labeled with [3H]oleic acid (2 μCi) and then switched to unlabeled diet until the fat body was used. This technique produced [3H]TAG-labeled fat body with the same specific activity in day 4 and 5 of the fifth instar insects and in all wandering stages. At the indicated developmental stages, [3H]TAG-labeled fat body (120 mg) was incubated in Grace's medium containing 1 mg/ml unlabeled larval Lp for 2 h and transfer studies were carried out as described above. Antiserum against purified LTP was obtained from a New Zealand White rabbit as described by Ryan et al. (21Ryan R.O. Senthilathipan K.R. Wells M.A. Law J.H. Facilitated diacylglycerol exchange between insect hemolymph lipophorins.J. Biol. Chem. 1988; 263: 14140-14145Abstract Full Text PDF PubMed Google Scholar). The IgG fraction was then purified using Affi-Gel Protein A and stored at −80°C. Protein concentration was determined by the Bradford assay (22Bradford M.M. A rapid and sensitive method for the quantitation of microgram quantities of proteins utilizing the principle of protein-dye binding.Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216391) Google Scholar) using BSA as the standard. Lipids were extracted according to Folch, Lees, and Sloane Stanley (20Folch J. Lees M. Sloane Stanley G.H. A simple method for isolation and purification of total lipids from animal tissues.J. Biol. Chem. 1957; 226: 497-509Abstract Full Text PDF PubMed Google Scholar) or Bligh and Dyer (23Bligh E.G. Dyer W.J. A rapid method of total lipid extraction and purification.Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (42865) Google Scholar). Lipid classes were separated by TLC on silica gel using hexane-ethyl ether-formic acid (70:30:3, v/v/v) as a solvent system (24Henderson R.J. Tocher D.R. Thin-layer chromatography.in: Hamilton R.J. Hamilton S. Lipid Analysis, A Practical Approach. Oxford University Press, New York1992: 65-111Google Scholar). Statistical tests were performed using GraphPad Prism version 3.00 for Windows (GraphPad Software, San Diego, CA). The results are expressed as means ± SEM. P < 0.05 was considered a significant difference between means. Figure 1shows the time course of lipid transfer when labeled Lp was incubated with larval M. sexta fat body. Over a 4 h incubation period, a significant amount of the radioactivity recovered in DAG and TAG was transferred from [3H]DAG-Lp to fat body. In contrast, a negligible amount of radioactivity was found in fat body lipids when incubations were performed at 4°C. Trypan blue exclusion tests performed during the incubation time showed that tissue integrity and cellular viability were maintained. As shown in Fig. 2, treatment of fat body with anti-LTP antibody before its incubation with [3H]DAG-Lp resulted in a significant inhibition of the transfer of labeled DAG from Lp to fat body. The decrease of radioactivity in fat body DAG and fat body TAG amounted to ∼40% and 60%, respectively, compared with controls. This incomplete inhibition of lipid transfer, in contrast to the results in larval midgut (12Canavoso L.E. Wells M.A. Role of lipid transport particle in lipid delivery of diacylglycerol from midgut to lipophorin in larval Manduca sexta.Insect Biochem. Mol. Biol. 2001; 31: 783-790Crossref PubMed Scopus (39) Google Scholar), did not change when increasing amounts of anti-LTP antibody up to 6 μg IgG/μl were tested. No inhibition on [3H]DAG transfer from Lp to fat body was observed if the tissue was preincubated with nonimmune rabbit serum (Fig. 2A), showing that the inhibitory effect of anti-LTP antibody was specific. When fat bodies preincubated with anti-LTP antibodies were transferred to a medium containing both [3H]DAG-Lp and LTP, the amount of lipid transferred to the tissue was restored to control values (Fig. 2B). It was also observed that the amount of [3H]DAG transferred to fat body after pretreatment with anti-LTP antibody increased with increasing concentrations of LTP up to 80 μg of LTP per assay, a concentration corresponding to the physiological levels for LTP, which were reported to be between 50 and 80 μg/ml during the fifth instar (10Van Heusden M.C. Yepiz-Plascencia G.M. Walker A.M. Law J.H. Manduca sexta lipid transfer particle: synthesis by fat body and occurrence in the hemolymph.Arch. Insect Biochem. Physiol. 1996; 31: 273-287Crossref PubMed Scopus (14) Google Scholar). The presence of a fat body membrane-bound lipase that could hydrolyze DAG-Lp into fatty acids and glycerol, which in turn would be taken up by the tissue and resynthesized into TAG, could account for the fact that anti-LTP antibodies did not completely inhibit DAG transfer to larval fat body. To test this possibility, we prepared dual-labeled DAG-Lp with [14C]fatty acid- and [3H]glycerol-labeled moieties and incubated it with fat body in the presence and absence of unlabeled glycerol. The rationale for this experiment was that if significant hydrolysis of DAG occurred because of the action of a membrane-bound lipase, then the presence of unlabeled glycerol would increase the 14C/3H ratio of the acylglycerols in the fat body. In fact, the presence of unlabeled glycerol had no effect on the 14C/3H ratio of acylglycerols in the fat body (Fig. 3). These results strongly suggest that DAG-Lp enters the cell without being hydrolyzed. Another mechanism that could account for the incomplete inhibition of DAG-Lp transfer in the presence of anti-LTP antibody is the possibility that Lp itself is endocytosed. We tested the effect of two endocytic inhibitors, ammonium chloride and chloroquine, on the transfer of lipid from Lp to fat body (Fig. 4). Chloroquine had no effect at any concentration tested, and ammonium chloride only inhibited at very high doses (100 mM), which is much higher than usually used in these studies. Thus, neither a lipoprotein lipase nor endocytosis could account for LTP-independent DAG-Lp transfer to the fat body. To test the possibility that DAG fat body represents a pool attached to the external surface of the fat body, even after Lp was washed away, we treated the fat body with lipase. The rationale for these experiments was that if the lipid remained on the external surface of the fat body, it would be accessible to a lipase added to the medium; on the other hand, if the lipid had been transferred into the tissue, it would not be accessible to lipase. As judged by the production of oleic acid after lipase addition, a significant fraction of the lipids present in the fat body sample after extensive washing, and with or without treatment with anti-LTP antibody, were still on the external surface of the fat body (Fig. 5). Our results indicate that added lipase was able to hydrolyze DAG in the anti-LTP antibody-insensitive pool into free fatty acid, which in turn was captured by BSA in the medium. In addition, DAG in this pool was being supplied from the hydrolysis of fat body TAG, which was decreased by 75%. Thus, we concluded that the majority of the 40% of the lipid bound to the fat body after anti-LTP treatment represents lipid present on the external surface of the fat body. Previously, we documented the presence of a Lp receptor on fat body of M. sexta (25Tsuchida K. Wells M.A. Isolation and characterization of a lipoprotein receptor from the fat body of an insect, Manduca sexta.J. Biol. Chem. 1990; 265: 5761-5767Abstract Full Text PDF PubMed Google Scholar) and that binding of Lp to its receptor is calcium-dependent. As shown in Fig. 6, neither Ca2+ nor EDTA in the incubation medium had a significant effect on lipid transfer. The transfer of lipid occurs best below pH 6.0 (Fig. 7), although the pH of hemolymph is 6.5. In addition, suramin, which inhibits the binding of Lp to its receptor (25Tsuchida K. Wells M.A. Isolation and characterization of a lipoprotein receptor from the fat body of an insect, Manduca sexta.J. Biol. Chem. 1990; 265: 5761-5767Abstract Full Text PDF PubMed Google Scholar) and binds to Lp, rendering it unavailable for binding (26Gondim K.C. Wells M.A. Characterization of lipophorin binding to the midgut of larval Manduca sexta.Insect Biochem. Mol. Biol. 2000; 30: 405-423Crossref PubMed Scopus (29) Google Scholar), also inhibited the transfer of DAG from Lp to fat body (Fig. 8). Taken together, these results are consistent with the suggestion that both LTP and a Lp receptor are involved in lipid transfer to the fat body.Fig. 7Effect of pH on [3H]DAG transfer. Labeled Lp was dialyzed into a buffer containing MES (0.01 M), MOPS (0.01 M), Trizma base (0.5 mM), and NaCl (0.15 M) of different pH values between 5.0 and 7.5. Labeled Lp (1.5 mg/ml) was incubated with fat body for 2 h, and transfer studies were carried out as described in Materials and Methods. Results are expressed as total dpm ± SEM (n = 3–4).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 8Effect of suramin on [3H]DAG transfer. Fat body was preincubated in 5 mM suramin for 30 min and then washed extensively with Grace's medium before adding [3H]DAG-Lp (1.5 mg/ml). After incubation for 2 h, the fat body was washed and lipids were analyzed as described for Fig. 1. Results are expressed as percentage DAG transferred ± SEM (n = 3–4).View Large Image Figure ViewerDownload Hi-res image Download (PPT) To determine the role of LTP and Lp receptor in the reverse transfer of DAG from the larval and wandering stages to Lp, we incubated [3H]DAG-labeled fat body tissues with larval Lp and determined the amount of [3H]DAG transferred to Lp (Fig. 9). The transfer of DAG between Lp and fat body is bidirectional. This occurs with fat body from either feeding-stage larvae or wandering-stage larvae. However, the uptake of DAG from Lp to the fat body was 3-fold higher than in the reverse direction, confirming the function of the fat body as a storage organ. In addition, lipid transfer in both directions is inhibited by anti-LTP antibodies and by suramin, suggesting that the basic process may be the same. Figure 10depicts the changes in the extent of DAG transfer from and to Lp at different developmental stages. The bidirectional transfer of lipid in this in vitro system decreased significantly from the feeding larval stage until the second day of wandering. The percentage decrease in the amount of DAG transferred from fat body to Lp and from Lp to fat body ranged from 7% to 60% and from 20% to 50% in the wandering stages compared with day 4 of fifth instar, respectively. Vertebrate systems contain several types of lipoproteins that deliver their neutral lipids to target tissues by a combination of lipoprotein lipase-mediated lipolysis (chylomicrons and VLDL) or endocytosis and degradation of the whole particle (LDL and chylomicron remnants) [reviewed in ref. (2Canavoso L.E. Wells M.A. Metabolic pathways for diacylglycerol biosynthesis and release in the midgut of larval Manduca sexta.Insect Biochem. Mol. Biol. 2000; 30: 1173-1180Crossref PubMed Scopus (30) Google Scholar)]. In the more versatile insect system, the same basic Lp particle carries a wide variety of lipids and selectively delivers specific lipids to specific tissues, e.g., cholesterol to oocytes (13Jouni Z.E. Takada N. Gazard J. Maekawa H. Wells M.A. Tsuchida K. Transfer of cholesterol and diacylglycerol from lipophorin to Bombyx mori oocytes in vitro: role of the lipid transfer particle.Insect Biochem. Mol. Biol. 2003; 33: 145-153Crossref PubMed Scopus (28) Google Schol
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