Fatty acid incorporation by Rhodnius prolixus midgut

[(14)C]Oleic acid injected into the hemocoel of Rhodnius prolixus females was shown to rapidly associate with lipophorin particles. Half of the lipophorin-associated [(14)C]oleic acid was transferred in about 5 min to different organs, but the midgut was the main organ to take it up on day 10 after a blood meal. The rate of [(14)C]oleic acid incorporation by the midgut was high up to 15 min after injection and then declined. The [(14)C]oleic acid incorporated by the midgut was found in phospholipids (58.6%) and neutral lipids (37.4%). The midgut capacity to incorporate [(14)C]oleic acid varied on different days after a meal: it increased up to day 10 and then decreased. The fate of the [(14)C]lipids synthesized by the midgut was followed and it was observed that 10 days after feeding diacylglycerol was the main lipid released to hemolymph and that most of phospholipids and triacylglycerols remained associated with the midgut. The metabolism of free fatty acids in Rhodnius prolixus females is discussed in the context of major biological events that follow a blood meal such as digestion and oogenesis.     

In vivo and in vitro loading of lipid by artificially lipid-depleted lipophorins: evidence for the role of lipophorin as a reusable lipid shuttle.

Lipid transport in the hemolymph of Manduca sexta is facilitated by a high density lipophorin in the resting adult insect (HDLp-A, d approximately 1.109 g/ml) and by a low density lipophorin during flight (LDLp, d approximately 1.060 g/ml). Lipophorin presumably shuttles different lipids between sites of uptake or storage, and sites of utilization. In order to shuttle lipid, a lipid-depleted lipophorin should be able to reload with lipid. To test this hypothesis, we used HDLp-A particles that were artificially depleted of either phospholipid (d approximately 1.118 g/ml) or diacylglycerol (d approximately 1.187 g/ml) and subsequently radiolabeled in their protein moiety. Upon injection into adult moths, both particles shifted their density to that of native HDLp-A, indicating lipid loading. Also, upon subsequent injection of adipokinetic hormone, both particles shifted to a lower density (d approximately 1.060 g/ml) indicating diacylglycerol loading and conversion to LDLp. Both phospholipid and diacylglycerol loading were also studied using an in vitro system. The lipid-depleted particles were incubated with fat body that had been radiolabeled in either the phospholipid or the triacylglycerol fraction. Transfer of radiolabeled phospholipid and diacylglycerol from fat body to lipophorin was observed. During diacylglycerol loading, apoLp-III associated with lipophorin, whereas phospholipid loading occurred in the absence of apoLp-III. The results show the ability of lipid-depleted lipophorins to reload with lipid and therefore reaffirm the role of lipophorin as a reusable lipid shuttle.     

Loading of lipophorin particles with phospholipids at the midgut of Rhodnius prolixus

32P-Labelled midguts (32P-midguts) of Rhodnius prolixus females were incubated in the presence of nonradioactive purified lipophorin and the release of radioactivity to the medium was analysed. The radioactivity found in the medium was associated with lipophorin phospholipids. When the 32P-midguts were incubated in the absence of lipophorin, no 32P-phospholipids were found in the medium. Comparative analysis by thin-layer chromatography of 32P-phospholipids derived from metabolically labelled 32P-midgut or lipophorin particles after incubation with 32P-midgut showed some differences, revealing a possible selectivity in the process of phospholipids transfer. The transfer of phospholipids to lipophorin was linear with time up to 45 min, was saturable with respect to the concentration of lipophorin, and was half-maximal at about 5 mg/ml. The binding of 32P-lipophorin to the midgut at 0 degrees C reached the equilibrium at about 1 h of incubation. The binding of 32P-lipophorin was inhibited by an excess of nonradioactive lipophorin, which suggests a specific receptor for lipophorin. The capacity of midguts and fat bodies to transfer phospholipids to lipophorin varied during the days following the meal. When lipophorin enzymatically depleted of phospholipids by treatment with phospholipase A2 was incubated with 32P-midguts, the same amount of phospholipids was transferred, indicating a net gain of phospholipids by the particle.     

Transfer of phospholipids from fat body to lipophorin in Rhodnius prolixus

32P-Labeled fat bodies (32P-fat bodies) of Rhodnius prolixus females were incubated in the presence of non radioactive purified lipophorin and the release of radioactivity to the medium was analysed to answer the question of whether lipophorin is a reusable shuttle for phospholipids. The radioactivity found in the medium was associated with lipophorin phospholipids. When the 32P-fat bodies were incubated in the absence of lipophorin, only a small amount of radioactivity was released and it was not associated with lipophorin, indicating that there was no release of pre-labeled 32P-lipophorin by the tissue. Analysis of 32P-phospholipids transferred from fat bodies to the lipophorin particles by thin-layer chromatography revealed a predominance of phosphatidylethanolamine and phosphatidylcholine, with minor amounts of phosphatidylserine, phosphatidylinositol, and sphingomyelin. The transfer of phospholipids to lipophorin was linear with time up to 45 min and the process was inhibited at low temperature and by the metabolic inhibitors azide and fluoride. The transfer of phospholipids from the fat bodies to lipophorin was saturable with respect to the concentration of lipophorin, which was half-maximal at about 8 mg/ml. A directional movement of phospholipids from the fat body to lipophorin was observed. The net gain of phospholipids in 2 h of incubation with fat body was 8.54 nmol per insect, which corresponds to 6.69% of increase in the lipophorin phospholipid content. The rate of 32P-phospholipid transfer from fat body to lipophorin particles varied during the days after a blood meal increasing up to day 10 and then decreasing in parallel with the process of oogenesis.     

Loading of lipophorin particles with phospholipids at the midgut of Rhodnius prolixus

³²P-Labelled midguts (³²P-midguts) of Rhodnius prolixus females were incubated in the presence of nonradioactive purified lipophorin and the release of radioactivity to the medium was analysed. The radioactivity found in the medium was associated with lipophorin phospholipids. When the ³²P-midguts were incubated in the absence of lipophorin, no ³²P-phospholipids were found in the medium. Comparative analysis by thin-layer chromatography of ³²P-phospholipids derived from metabolically labelled ³²P-midgut or lipophorin particles after incubation with ³²P-midgut showed some differences, revealing a possible selectivity in the process of phospholipids transfer. The transfer of phospholipids to lipophorin was linear with time up to 45 min, was saturable with respect to the concentration of lipophorin, and was half-maximal at about 5 mg/ml. The binding of ³²P-lipophorin to the midgut at O°C reached the equilibrium at about 1 h of incubation. The binding of ³²P-lipophorin was inhibited by an excess of nonradioactive lipophorin, which suggests a specific receptor for lipophorin. The capacity of midguts and fat bodies to transfer phospholipids to lipophorin varied during the days following the meal. When lipophorin enzymatically depleted of phospholipids by treatment with phospholipase A² was incubated with ³²P-midguts, the same amount of phospholipids was transferred, indicating a net gain of phospholipids by the particle. © 1995 Wiley-Liss, Inc.     

Lipophorin of the larval honeybee, Apis mellifera L

Most insects have a major lipoprotein species in the blood (hemolymph) that serves to transport fat from the midgut to the storage depots in fat body cells and from the fat body to peripheral tissues. The generic name lipophorin is used for this lipoprotein. In larvae of the honeybee, Apis mellifera, a lipophorin has been found with properties that correlate well with those of the only other lipophorin reported for an immature insect, that of the tobacco hornworm, Manduca sexta. The honeybee lipophorin (Mr = 530,000) has a density of 1.13 g/ml, contains approximately 41% lipid and 59% protein, and contains two apoproteins, apoLp-I, Mr = 250,000 and apoLp-II, Mr = 80,000, both of which are glycosylated. The lipids consist predominantly of polar lipids, of which phospholipids and diacylglycerols represent 60% of the total. When the intact lipophorin is treated with trypsin, apoLp-I is rapidly proteolyzed, while apoLp-II is resistant, indicating a difference in exposure of the two apoproteins to the aqueous environment. Honeybee apoLp-II cross-reacts with antibodies to M. sexta apoLp-II, but not to anti-M. sexta apoLp-I. No cross-reactivity of honeybee apoLp-I to anti-M. sexta apoLp-I was observed.     

β-chain of ATP synthase as a lipophorin binding protein and its role in lipid transfer in the midgut of Panstrongylus megistus (Hemiptera: Reduviidae)

Lipophorin, the main lipoprotein in the circulation of the insects, cycles among peripheral tissues to exchange its lipid cargo at the plasma membrane of target cells, without synthesis or degradation of its apolipoprotein matrix. Currently, there are few characterized candidates supporting the functioning of the docking mechanism of lipophorin-mediated lipid transfer. In this work we combined ligand blotting assays and tandem mass spectrometry to characterize proteins with the property to bind lipophorin at the midgut membrane of Panstrongylus megistus, a vector of Chagas' disease. We further evaluated the role of lipophorin binding proteins in the transfer of lipids between the midgut and lipophorin. The β subunit of the ATP synthase complex (β-ATPase) was identified as a lipophorin binding protein. β-ATPase was detected in enriched midgut membrane preparations free of mitochondria. It was shown that β-ATPase partially co-localizes with lipophorin at the plasma membrane of isolated enterocytes and in the sub-epithelial region of the midgut tissue. The interaction of endogenous lipophorin and β-ATPase was also demonstrated by co-immunoprecipitation assays. Blocking of β-ATPase significantly diminished the binding of lipophorin to the isolated enterocytes and to the midgut tissue. In vivo assays injecting the β-ATPase antibody significantly reduced the transfer of [³H]-diacylglycerol from the midgut to the hemolymph in insects fed with [9,10-³H]-oleic acid, supporting the involvement of lipophorin-β-ATPase association in the transfer of lipids. In addition, the β-ATPase antibody partially impaired the transfer of fatty acids from lipophorin to the midgut, a less important route of lipid delivery to this tissue. Taken together, the findings strongly suggest that β-ATPase plays a role as a docking lipophorin receptor at the midgut of P. megistus.     

An insect lipid transfer particle promotes lipid loading from fat body to lipoprotein

The role of Manduca sexta lipid transfer particle (LTP) in the transport of lipid from fat body to lipophorin was investigated in vitro. Fat body that contained radiolabeled lipid was incubated with either high density lipophorin or low density lipophorin, and it was shown that lipid was transferred from fat body to lipophorins. The transfer of diacylglycerol was blocked by preincubating fat body with LTP antibody. Furthermore, transfer was restored by the addition of LTP, indicating that LTP promotes the transfer of lipid from fat body to lipophorins. Using lipophorins radio-labeled in their lipid moiety, transfer of lipid from lipophorin to fat body was demonstrated. This transfer was not mediated by LTP. The adipokinetic hormone induced diacylglycerol mobilization from the fat body and the concomitant interconversion of high density lipophorin to low density lipophorin were performed in vitro and were shown to require the presence of LTP.     

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.     

A function for plasma membrane reticular systems

The basal surface in transporting epithelia is infolded in a way that encourages the formation of standing gradients. Many insect cells have a similar infolded reticular system (RS) although they are clearly not transporting epithelia. These cells are like one another metabolically in that they sequester lipid from hemolymph lipophorins (lipid transporting proteins). Dietary lipids enter the hemolymph from the midgut RS which may be an adaptation for lipophorin loading. The plasma membrane reticular system of tissues metabolizing lipids (fat body, wax glands, oenocytes, lenticles) may be an adaptation for lipophorin reception and unloading. Cationic ferritin (pI 8.5) shows all RSs are covered by a lamina functioning as a negatively charged sieve. The basal plasma membrane leading to the RS is also negatively charged. The RS is a container with charged entrances that would be expected to affect the composition of the contents. Midgut cells release lipid particles into their RS. The particles are positively charged since in tracer studies they associate with anionic but not cationic ferritin. Lipophorins are anionic. The electrostatic binding of lipid to lipophorin would make it less anionic and more likely to leave the RS when loaded, thus carrying lipid to the hemolymph. Conversely, at the destination RS, loaded lipophorin would penetrate more easily than unloaded. A change in charge with unloading would be expected to alter the equilibrium between entering and leaving lipophorin, causing protein concentration in the RS of lipid receiving tissues as has been observed in the fat body. Reticular systems may thus be reaction vessels for interactions between carrier proteins and their load.     

Lipid transfer particle mediates the delivery of diacylglycerol from lipophorin to fat body in larval Manduca sexta

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 [(3)H]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 [(3)H]DAG transfer from lipophorin. Treatment of [(3)H]DAG-labeled fat body with lipase significantly reduced the amount of [(3)H]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.     

Locust adipokinetic hormone stimulates lipid mobilization in Manduca sexta

Adipokinetic hormone, a decapeptide isolated from the locust, stimulates mobilization of diacylglycerols from the locust fat body and loading of the lipid transport protein, lipophorin. Injection of the synthetic locust adipokinetic hormone into a sphinx moth, Manduca sexta, causes lipid loading of lipophorin. The lipophorin decreases in density from 1.11 to 1.06 g/ml, and a soluble protein from the hemolymph (apolipophorin III) associates with the lipophorin particle. Administration of intermediate doses of hormone indicates that lipophorin is converted directly to the low density form; no appreciable amounts of intermediate density particles are formed.     

Characterization of cholesterol transport from midgut to fat body in Manduca sexta larvae

Using in vitro methods, we investigated the transfer of cholesterol from larval Manduca sexta midgut to the hemolymph lipoprotein, lipophorin, and the transfer of cholesterol from lipophorin to larval fat body. In the midgut, transfer of free cholesterol shows saturation kinetics, but the apparent Km is higher than the measured Kd for the midgut lipophorin-receptor complex. In addition, the transfer is unaffected by suramin, which binds to the receptor and inhibits lipophorin binding, and by antibodies to the lipid transfer particle, which is required for export of diacylglycerol from the midgut to lipophorin. In the fat body, transfer of free cholesterol also shows saturation kinetics, and the apparent Km is higher than the measured Kd for the fat body lipophorin-receptor complex. Suramin and anti-lipid transfer particle antibodies exert only a small (20%) inhibitory effect. In both tissues it seems that the most likely mode of cholesterol transfer is via aqueous diffusion, which is also an important mechanism in vertebrate cells. Based on these results, we propose that cholesterol homeostasis in larval M. sexta is maintained by a mass action mechanism in which cholesterol is freely transferred between lipophorin and tissues depending on the needs of the tissues. This simple mechanism is ideally suited to insects, which can neither make cholesterol nor internalize lipophorin, the two mechanisms that vertebrate cells use to control their cholesterol content.     

In vitro release of lipophorin from the fat body of nondiapause and diapause larvae of the Southwestern corn borer,Diatraea grandiosella

The release of lipophorin and total protein was examined from the fat body of nondiapause and diapause larvae of the southwestern corn borer, Diatraea grandiosella, incubated in vitro in Grace's medium. The characteristics of the released lipophorin were compared to those of the high-density lipophorin present in the hemolymph of nondiapause and diapause larvae. Over a 4 h incubation period, the fat body of nondiapause larvae released about 1.5 times more total protein and 2 times more lipophorin per mg dry weight than did that of diapause larvae. Lipophorin isolated from the medium in which fat bodies of nondiapause and diapause larvae had been incubated and from the plasma of nondiapause and diapause larvae had similar mean densities of 1.115, 1.112, 1.117 and 1.119 g/ml, respectively. Although the lipid classes detected in lipophorin isolated from the fat body incubation medium and hemolymph were identical, more polar lipids and less diacylglycerol were associated with lipophorin isolated from fat body incubation medium then were associated with lipophorin isolated from the hemolymph. Sterols accounted for about 11% of the total lipids of lipophorin isolated from the fat body incubation medium, whereas they accounted for about 20% of the total lipids of lipophorin from hemolymph. We conclude that the fat body of feeding nondiapause larvae and nonfeeding diapause larvae releases high-density lipophorin.     

Rhodnius prolixus LIPOPHORIN: LIPID COMPOSITION AND EFFECT OF HIGH TEMPERATURE ON PHYSIOLOGICAL ROLE

Lipophorin is a major lipoprotein that transports lipids in insects. In Rhodnius prolixus, it transports lipids from midgut and fat body to the oocytes. Analysis by thin‐layer chromatography and densitometry identified the major lipid classes present in the lipoprotein as diacylglycerol, hydrocarbons, cholesterol, and phospholipids (PLs), mainly phosphatidylethanolamine and phosphatidylcholine. The effect of preincubation at elevated temperatures on lipophorin capacity to deliver or receive lipids was studied. Transfer of PLs to the ovaries was only inhibited after preincubation of lipophorin at temperatures higher than 55°C. When it was pretreated at 75°C, maximal inhibition of phospholipid transfer was observed after 3‐min heating and no difference was observed after longer times, up to 60 min. The same activity was also obtained when lipophorin was heated for 20 min at 75°C at protein concentrations from 0.2 to 10 mg/ml. After preincubation at 55°C, the same rate of lipophorin loading with PLs at the fat body was still present, and 30% of the activity was observed at 75°C. The effect of temperature on lipophorin was also analyzed by turbidity and intrinsic fluorescence determinations. Turbidity of a lipophorin solution started to increase after preincubations at temperatures higher than 65°C. Emission fluorescence spectra were obtained for lipophorin, and the spectral area decreased after preincubations at 85°C or above. These data indicated no difference in the spectral center of mass at any tested temperature. Altogether, these results demonstrate that lipophorin from R. prolixus is very resistant to high temperatures.     

Lipophorin interaction with the midgut of Rhodnius prolixus: characterization and changes in binding capacity

Several classes of lipids are transported in insect hemolymph by lipophorin, a major hemolymphatic lipoprotein. The binding of lipophorin to the midgut of the hematophagous insect Rhodnius prolixus was characterized in a midgut membrane preparation, using purified lipophorin radiolabelled in protein moiety ((125)I-HDLp). Lipophorin specific binding to membranes achieved equilibrium after 30-40 min, was sensitive to pH, and was maximal at pH 7.0. In the presence of increasing concentrations of membrane protein, corresponding increases in lipophorin binding were observed. The specific binding of lipophorin to the membrane preparation was a saturable process, with K(d)=0.9+/-0.06 x 10(-7) M and a maximal binding capacity of 70+/-11 ng lipophorin/microg of membrane protein. Lipophorin binding did not depend on calcium, but it was affected by ionic strength and was inhibited in the presence of increasing salt concentrations. Suramin interfered with lipophorin binding to the midgut receptor, and it was abolished in the presence of 2 mM suramin, but at concentrations between 0.05 and 0.2 mM it was slightly increased. Condroitin 4-sulfate also affected lipophorin binding, which was reduced to 56% of control. Pre-incubation of the midgut membrane preparation with trypsin or at high temperature inhibited binding. Midgut capacity to bind lipophorin varied at different days after blood meal. It was highest at second day after feeding, and then gradually decreased.     

Conversion of human low density lipoprotein into a very low density lipoprotein-like particle in vitro.

The lipid substrate specificity of Manduca sexta lipid transfer particle (LTP) was examined in in vitro lipid transfer assays employing high density lipophorin and human low density lipoprotein (LDL) as donor/acceptor substrates. Unesterified cholesterol was found to exchange spontaneously between these substrate lipoproteins, and the extent of transfer/exchange was not affected by LTP. By contrast, transfer of labeled phosphatidylcholine and cholesteryl ester was dependent on LTP in a concentration-dependent manner. Facilitated phosphatidylcholine transfer occurred at a faster rate than facilitated cholesteryl ester transfer; this observation suggests that either LTP may have an inherent preference for polar lipids or the accessibility of specific lipids in the donor substrate particle influences their rate of transfer. The capacity of LDL to accept exogenous lipid from lipophorin was investigated by increasing the high density lipophorin:LDL ratio in transfer assays. At a 3:1 (protein) ratio in the presence of LTP, LDL became turbid (and aggregated LDL were observed by electron microscopy) indicating LDL has a finite capacity to accept exogenous lipid while maintaining an overall stable structure. When either isolated human non B very low density lipoprotein (VLDL) apoproteins or insect apolipophorin III (apoLp-III) were included in transfer experiments, the sample did not become turbid although lipid transfer proceeded to the same extent as in the absence of added apolipoprotein. The reduction in sample turbidity caused by exogenous apolipoprotein occurred in a concentration-dependent manner, suggesting that these proteins associate with the surface of LDL and stabilize the increment of lipid/water interface created by LTP-mediated net lipid transfer. The association of apolipoprotein with the surface of modified LDL was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, and scanning densitometry revealed that apoLp-III bound to the surface of LDL in a 1:14 apoB:apoLp-III molar ratio. Electron microscopy showed that apoLp-III-stabilized modified LDL particles have a larger diameter (29.2 +/- 2.6 nm) than that of control LDL (22.7 +/- 1.9 nm), consistent with the observed changes in particle density, lipid, and apolipoprotein content. Thus LTP-catalyzed vectorial lipid transfer can be used to introduce significant modifications into isolated LDL particles and provides a novel mechanism whereby VLDL-LDL interrelationships can be studied.     

Formation of lipid reserves in fat body and eggs of the yellow fever mosquito, Aedes aegypti

We examined the accumulation of lipids in adult females of the mosquito, Aedes aegypti. Females emerged with about 100 μg lipid in the fat body. With access to sugar water lipids increased over seven days to 300 μg. After a blood meal on day five, sugar-fed females accumulated 120-140 μg of lipids in their ovaries within 2 days. At the same time the lipid content of the fat body decreased by 100 μg, indicating transfer of lipids from fat body to oocytes. Experiments in which fat body lipids were prelabelled support this conclusion. Label was transferred to oocytes: in mature oocytes the specific radioactivity of lipids was 80% of the specific radioactivity of prelabeled fat body lipids. Components of blood meals are also used to synthesize oocyte lipids. Fat bodies of females starved for four days had only 27 μg of lipids left. When these females were given a blood meal, they matured oocytes, although the number of ooyctes was reduced and ovaries contained only half the amount of lipids found in ovaries of females which had first fed on sugar water. Fat body lipids of these females had only slightly increased to 36 μg. This demonstrates that female Ae. aegypti use sugar to synthesize lipids, but they can also use components of blood for this purpose.