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.     

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.     

Rhipicephalus sanguinius: localization of vitellogenin synthesis by immunological methods and electron microscopy

In ovipositing Rhipicephalus sanguinius (Latrelle), complete immunological identity existed between vitellogenin from the midgut, fat body, and hemolymph and vitellin from eggs. This supported the hypothesis that the same vitellogenin was synthesized by both the midgut and fat body, then released into the hemolymph and transported to the ovary. Vitellogenin was taken up unaltered by the oocytes during vitellogenesis to become vitellin. Antivitellogenin did not react with host (dog) hemoglobin. Transmission electron microscopy showed specialized cells with large amounts of rough endoplasmic reticulum, Golgi complexes, and secretory granules in the midgut and fat body of ovipositing females that were absent in the midgut and fat body of fed males. It is suggested that these cells synthesize vitellogenin.     

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.     

Changes in the titer of the female-predominant storage protein (81K) during larval and pupal development of the waxmoth,galleria mellonella

The levels of an 81K storage protein in the waxmoth, Galleria mellonella, were monitored during the course of development using rocket immunoelectrophoresis. During the fifth and sixth larval stadia, 81K protein levels increased during feeding and growth but sharply declined at each larval molt. During the fifth and sixth stadia hemolymph levels of the 81K protein increased to about 1 and 2.5 mg/ml, respectively, with no discernible differences between levels in males and females. Neither the fat body nor the remainder of the carcass contained the 81K protein, indicating that the accumulation of this protein during the intermolt period was exclusively in the hemolymph and redistribution of the 81K protein into other tissues does not occur at the final two larval molts. During the seventh (final) larval stadium the absolute quantities of the 81K protein increased from 23 μg per insect to over 1,600 μg in females and to 300 μg in males. The hemolymph concentration of the 81K protein reached 28 mg/ml in females and 6 mg/ml in males with only low levels found in the remaining tissues. Shortly after pupal apolysis, marked by eyespot retraction, the fat body in both sexes rapidly and quantitatively sequestered the 81K protein from the hemolymph. The 81K protein in the hemolymph of both males and females rapidly dropped to nearly zero concentration by pupation. The 81K storage protein remained localized in the fat body cells after uptake occurred, even though the fat body cells disaggregate and reaggregate during metamorphosis. During pharate adult development the 81K storage protein disappeared from the fat body without entering the hemolymph. At adult eclosion 81K was virtually absent from the tissues of both males and females.     

Site of synthesis,tissue distribution,and lipophorin transport of hydrocarbons in Blattella germanica (L.) nymphs

The site of hydrocarbon (HC) synthesis and the amount of HC in various tissues were investigated in relation to developmental stage in the last larval stadium of the German cockroach, Blattella germanica. Abdominal integument linearly incorporated [1-(14)C]propionate into HC for at least 6h in vitro, whereas other body parts synthesized little or no HC. The third through sixth abdominal sternites and tergites were the principal sites of synthesis. High rates of HC synthesis resulted in a fivefold increase in internal HC during the last stadium. We examined the distribution of HC in the hemolymph, fat body, and the developing imaginal cuticle. Hemolymph HC titer was relatively constant at approximately 8&mgr;g/&mgr;l. However, as hemolymph volume increased from 5 to 11&mgr;l in the first 4days of the last stadium, HC content increased and then remained stable the remainder of the stadium. Lipophorin, immunoprecipitated with adult lipophorin polyclonal antibodies, was the only HC carrier protein in nymphal hemolymph and its HC profile was identical to that of hemolymph and similar to that of the epicuticle. The concentration and total amount of hemolymph lipophorin increased until 3days before adult eclosion and declined immediately after ecdysis. The HC content of non-biosynthetic integument (legs, pronotum) doubled during formation of the imaginal cuticle, as did the HC content of sternites, which synthesize HC. HC content of fat body, however, increased threefold during the same period, suggesting that the fat body serves as a storage site for HC during cuticle formation. We conclude that in the last stadium HC is synthesized by abdominal oenocytes, loaded onto hemolymph lipophorin, and transported to fat body and both nymphal and imaginal cuticle. Hydrocarbons associate with the imaginal integument several days before eclosion.     

Synthesis and release of lipophorin in larvae of the southwestern corn borer,Diatraea grandiosella: An in vitro study

The source of the lipophorin present in the larval haemolymph of the southwestern corn borer, Diatraea grandiosella, was examined in vitro. Although lipophorin was shown to be one of several proteins released from cultured fat body and midgut, only fat body was shown to synthesize lipophorin. Fat body, incubated in a medium containing [³H]leucine, was shown to release radiolabelled lipophorin using immunoprecipitation. Similar studies using midguts incubated in a medium containing [³H]leucine did not reveal any synthesis of lipophorin. Lipophorin was isolated by density-gradient ultracentrifugation from media in which the fat bodies of about 600 diapausing larvae had been incubated for 4 hr. The isolated lipophorin had a peak density of 1.11 g/ml, and contained various lipids including diacylglycerol, triacylglycerol, sterol, hydrocarbon, free fatty acid, phosphatidyl choline, phosphatidyl ethanolamine and sphingomyelin.     

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.     

Changes of acid phosphatase content and activity in the fat body and the hemolymph of the flesh fly Neobellieria (sarcophaga) bullata during metamorphosis

Changes in the specific and total activity of the lysosomal marker enzyme acid phosphatase (Acph) and in the amount of enzyme protein were examined in the fat body and the hemolymph from the last larval molt to the larval-pupal apolysis. The specific activity showed minor changes during the last larval period. In contrast, the total activity of the enzyme was low during the feeding period and higher during the wandering stage and strikingly increased at the time of puparium formation. We purified a protein having para-nitrophenyl phosphate phosphatase (Acph) activity and raised antisera against it. The amount of Acph protein in the fat body and hemolymph was examined using an ELISA. The specific Acph content showed little variation, but the total amount of the enzyme protein showed a stepwise increase in both organs during last larval stage and was markedly elevated in the pupal stage in the fat body. In contrast, a considerable decrease in the amount of Acph protein was observed in the hemolymph during this period. These data were in agreement with immunohistochemical observations showing an accumulation of the enzyme protein in fat body cells during the prepupal stage with a concomitant disappearance of the enzyme from the hemolymph. Inhibition of ecdysteroid secretion by water stress prevented the changes both in total enzyme activity and in the amount of Acph protein. However, Acph protein content and enzyme activity could be restored when the water stress was followed by a 20-hydroxyecdysone (20-HE) treatment. Taken together, our data show that Acph is secreted by fat body cells into the hemolymph during the larval stage, where it is stored in an inactive form. Increase in the 20-HE titer at the end of last larval stage reverses this process, and the enzyme is taken up by the fat body cells, where it becomes activated and appears in auto- and heterophagic vacuoles. Arch. Insect Biochem. Physiol. 34:369–390, 1997. © 1997 Wiley-Liss, Inc.     

Dynamics of lipid accumulation by the fat body of Rhodnius prolixus: the involvement of lipophorin binding sites

In insects, lipids are stored in the fat body, mainly as triacylglycerol (TAG). In Rhodnius prolixus, a hematophagous hemipteran, lipids are accumulated after blood meal to be used later on. In adult females, at the second day after feeding, the amount of TAG was 57+/-17 microg/fat body, it increased almost five times and at fourth day it was 244+/-35 microg/fat body. TAG content remained constant until day 13, but it then decreased and, at day 20th it was very low (31+/-4.9 microg/fat body). Radiolabeled free fatty acid was used to follow lipid accumulation by the fat body, as it was previously shown that, in R. prolixus, injected free fatty acids associate with lipophorin, a major hemolymphatic lipoprotein. (3)H-palmitic acid was injected into the hemocoel of R. prolixus females. It disappeared from the hemolymph very rapidly, and radioactivity was incorporated by the fat body. Sixty minutes after injection, radioactivity in the fat body was found mainly in TAGs. The capacity of the fat body to incorporate fatty acids from the hemolymph varied according to the days after blood meal, and it was maximal around the fourth day. Lipophorin binding to specific sites in fat body membrane preparations also showed variation at different days. When membranes obtained from insects at the second, fifth and tenth days were compared, binding was highest at fifth day after feeding.     

Lipophorin density variation during oogenesis in Rhodnius prolixus

The density of lipophorin was determined in adult females of Rhodnius prolixus on different days after a meal. Several populations of lipophorins, differing in density but always in the range of HDL, were found in the hemolymph. The density of the major population was analyzed and a complex profile of density variation was found associated with the principal metabolic events in these insects digestion and oogenesis. During the initial three days after the blood meal, with the onset of the digestive process, the density of lipophorin decreased from 1.1185 g/l to 1.1095 g/l, associated with the transfer of lipids from midgut to the lipophorin particles. During the period of intense vitellogenesis and lipid uptake by the ovary, the lipophorin density started to increase and reached the value, 1.1322 g/l, and remained stable up to the end of oogenesis. As soon as the requirement of lipids to build up the oocytes ceased, the density of lipophorin decreased to its initial value associated with the transfer of lipids from fat body to lipophorin. Soon after the blood meal the midgut was the main source of lipids capable of replenishing the lipophorin particles, while the fat body assumed this function during the succeeding days and reached its maximum capacity around day 10, as estimated by the rate of lipid transfer. The principal lipids transferred were phospholipids and diacylglycerols. Except in the protein/lipid ratio no major changes were observed among different lipids isolated from lipophoin of different densities. Arch. Insect Biochem. Physiol. 35:301-313, 1997.© 1997 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.     

Venom of Euplectrus separatae causes hyperlipidemia by lysis of host fat body cells

Although the lepidopteran larva Pseudaletia separata is attacked by the gregarious ectoparasitoid Euplectrus separatae, it continues to feed and grow. Lipid concentration in the hemolymph of the parasitized host was higher than that of the nonparasitized host from 3 to 8 days after parasitization. Artificial injection of parasitoid venom also elevated lipid concentration in the host hemolymph. One day after venom injection the host's fat body contained many lipid particles, but most of the lipid particles disappeared 7 days later. Light microscopy and transmission electron microscopy showed the lipid particles leaving the fat body cells as a result of the lysis of the fat body cells. These results suggest that the venom elevated the lipid concentration in the host hemolymph by provoking the release of lipid particles from the fat body. Though most of the lipid particles were freely floating in the host hemolymph, a portion of the released lipid particles were phagocytized by hemocytes. The amount of lipid that was loaded to lipophorin in the hemolymph of the venom-injected host was measured, but it was not sufficient to explain the high lipid titer in the hemolymph of parasitized and venom-injected host larvae. The fact that parasitoid larva consumed many hemocytes as evidenced by their presence in the midgut supported the hypothesis that the parasitoid larvae fed on the host hemolymph containing the free lipid particles, the hemocytes phagocytizing the lipid particles, and the lipid-loaded lipophorin. The possibility of the venom contribution to the disruption of the intercellular matrix was examined. The venom showed high activity of matrix metalloproteinase (MMP), especially when it was mixed with the hemolymph of non-parasitized 5th instar larvae. We suggest that the MMP in the venom was activated by some components of the host hemolymph. On the other hand, the venom mixed with hemolymph could not decompose gelatin on zymography, suggesting that the venom-MMP is a different type from gelatinase. Activity of phospholipases A(2), B, C and hyaluronidase were measured with agar plates. High activities of phospholipase B and hyaluronidase were detected. These results suggest that the venom-MMP initially attacked the specific site of the intercellular-matrix of the fat body, and then the hyaluronidase and the phospholipase B cause lysis of the fat body cell, allowing lipid particles to be released into the host hemolymph.     

Proline can be utilized as an energy substrate during flight of Aedes aegypti females

In order to determine whether proline can be utilized as fuel during flight of Aedes aegypti, proline, alanine, and glutamine concentrations were monitored at 0, 30 and 60 min after flight using sugar-fed males and females, and blood meal-fed females. In sugar-fed and blood meal-fed females, flight lead to a significant decrease in proline and a significant increase in glutamine concentration in both hemolymph and thorax. Only during flight after a blood meal was a significant increase in the alanine concentration observed in hemolymph. After flight, the proline alanine and glutamine levels in the hemolymph and thorax from males did not change significantly. In addition, activities of enzymes related to amino acid metabolism were assayed in homogenates of cephalothorax and thorax from both sexes, and in fat body and midgut from females. In both sexes, the activities of all the enzymes studied were significantly higher in thorax than in cephalothorax. The levels of the enzymes involved in proline oxidation were higher in thorax than in fat body and midgut. These results suggest that proline can be used as an energy substrate for flight muscle of Ae. aegypti females. However, the elevation in glutamine levels observed in hemolymph and thorax after flight has not been reported in other insects that fuel flight using proline and may suggest an additional mechanism for shuttling ammonia between flight muscle and fat body is present in mosquitoes.     

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.     

Changes in lipoprotein composition during larval-pupal metamorphosis of an insect, Manduca sexta

During the transition from the last feeding larval stage to the pupal stage of the tobacco hornworm, Manduca sexta, significant changes occur in the properties of lipophorin, the major hemolymph lipoprotein. Within the first 24 h after cessation of feeding, the larval lipophorin (HDLp-L) is first converted to a higher density form (HDLp-W2) and then HDLp-W2 is converted to a lower density form (HDLp-W1). HDLp-W1 remains in the hemolymph until pupation, when another form, HDLp-P, with a density between HDLp-W1 and HDLp-L, is present. Although all the lipophorins contain identical apoproteins, they differ in lipid content and composition; the differences in density being primarily related to diacylglycerol content. The conversion of HDLp-L to HDLp-W1 is accompanied by a loss of hydrocarbon and uptake of carotenes. These latter changes in lipophorin composition reflect alterations in cuticular lipid composition. HDLp-L was radiolabeled in the apoproteins by injecting animals with 3H-amino acids early in the last larval stage. Subsequently HDLp-L was isolated at the end of the larval stage, HDLp-W2 and HDLp-W1 were isolated during the wandering stage, and HDLp-P was isolated after pupation. The specific activity of the apoproteins in the four lipophorins was not significantly different, suggesting that the observed alterations in lipophorin properties do not require synthesis of new apoproteins but result from retailoring the lipid composition of preexisting molecules. Examination of the hemolymph of individual animals during these transitions showed that only one species of lipoprotein was present, never a mixture of two or more species. These observations suggest that the lipoprotein conversions are precisely timed and that lipoprotein metabolism during larval development and pupation cannot be considered a static process. The unique finding of these studies was that synthesis of lipophorin apoproteins proceeds actively during the first part of the fifth instar but then ceases and does not recommence during the wandering or early pupal stages.     

Manduca sexta lipid transfer particle: Synthesis by fat body and occurrence in hemolymph

Lipid transfer particle (LTP) is present in hemolymph of the tobacco hornworm Manduca sexta. Biosynthesis of LTP, occurrence in hemolymph, and the role of LTP-apoproteins in the lipid transfer reaction were investigated using antibodies specific for LTP or for each of the apoproteins. In vitro protein synthesis followed by immunoprecipitation demonstrated that LTP is synthesized by the fat body and secreted into the medium. In contrast to apolipophorin III, an exchangeable apoprotein of lipophorin (the major lipid transport protein in hemolymph), apoLTP-III could not be detected free in hemolymph. LTP concentrations in the hemolymph were measured by a sandwich ELISA using a mouse monoclonal antibody against apoLTP-III as capturing antibody and rabbit polyclonal antibody against apoLTP-I as detecting antibody. LTP concentration increased during the late fifth instar larval stage, followed by a decrease in the wandering stage. Subsequently, LTP concentrations were strongly increased in hemolymph of adult moths. The role of the three apoproteins of LTP in the lipid transfer reaction was analyzed using apoprotein-specific antibodies. All three, apoLTP-I, -II, and -III, appeared to be important for lipid transfer activity, as shown by inhibition of lipid transfer by antibodies specific for each of the three apoproteins. © 1996 Wiley-Liss, Inc.     

Lipophorin in the larval and adult stages of Musca domestica

The major Musca domestica hemolymph lipoprotein, lipophorin, was purified from larval and from adult animals. The housefly lipophorin is composed of two apoproteins, apolipophorin I (M_r ∽ 253,000) and apolipophorin II (M_r ∽ 85,000). The lipophorin contains about 3.9% carbohydrates and reacts positively with concanavalin A. The density of larval lipophorin is equal to 1.152 g/ml and of adult lipophorin to 1.106 g/ml. The amount of lipophorin per animal increases during the larval stage, is constant during pupal stage, and suffers a great reduction at the pharate adult stage. The amount of lipophorin remains stable during the whole first gonotrophic cycle of the housefly. Lipophorin is not detected in the eggs of this insect.