Lipoproteins in Drosophila melanogaster-Assembly, Function, and Influence on Tissue Lipid Composition

Interorgan lipid transport occurs via lipoproteins, and altered lipoprotein levels correlate with metabolic disease. However, precisely how lipoproteins affect tissue lipid composition has not been comprehensively analyzed. Here, we identify the major lipoproteins of Drosophila melanogaster and use genetics and mass spectrometry to study their assembly, interorgan trafficking, and influence on tissue lipids. The apoB-family lipoprotein Lipophorin (Lpp) is the major hemolymph lipid carrier. It is produced as a phospholipid-rich particle by the fat body, and its secretion requires Microsomal Triglyceride Transfer Protein (MTP). Lpp acquires sterols and most diacylglycerol (DAG) at the gut via Lipid Transfer Particle (LTP), another fat body-derived apoB-family lipoprotein. The gut, like the fat body, is a lipogenic organ, incorporating both de novo-synthesized and dietary fatty acids into DAG for export. We identify distinct requirements for LTP and Lpp-dependent lipid mobilization in contributing to the neutral and polar lipid composition of the brain and wing imaginal disc. These studies define major routes of interorgan lipid transport in Drosophila and uncover surprising tissue-specific differences in lipoprotein lipid utilization.     

Isolation and characterization of a lipoprotein receptor from the fat body of an insect, Manduca sexta

A lipoprotein receptor has been purified from the fat body of Manduca sexta larvae. The purification involves solubilization of membrane proteins in detergent, DEAE-, and hydroxyapatite chromatography, affinity chromatography on a concanavalin A column, and affinity chromatography on a lipoprotein-Sepharose column. An overall purification of 220-fold from the solubilized membranes was achieved. The receptor has an apparent molecular mass of 120 kDa. The receptor has an absolute requirement for Ca2+ and is inhibited by Suramin. The pH optimum of the receptor is 6.5, which is near the pH of the hemolymph. Binding data indicate a single high affinity binding site with a Kd = 4.1 +/- 0.19 x 10(-8) M as measured with the lipoprotein isolated from larval hemolymph. The major neutral lipid carried by insect lipoproteins is diacylglycerol, and it was shown that the affinity of the receptor for lipoprotein ligands correlates with their diacylglycerol content. It is proposed that the decrease in affinity of the receptor for lipoproteins depleted of diacylglycerol plays a key role in facilitating the transport of diacylglycerol from the midgut to the fat body during the larval feeding period. The insect receptor has some properties which are similar to those of vertebrate lipoprotein receptors, viz. molecular weight, requirement for Ca2+, and inhibition by Suramin. However, the insect receptor does not bind human low density lipoprotein.     

Lipid uptake by insect oocytes

Approximately 30-40% of the dry weight of an insect egg consists of lipid, mostly triacylglycerol (TAG). Although this lipid is essential for the energy needed by the developing embryo, little is known about the mechanism that leads to the accumulation of TAG in the insect egg. Insect oocytes can readily synthesize TAG from free fatty acids (FFAs) and glycerol, however, de novo synthesis of FAs by the oocyte is marginal. Hence, FAs have to be imported from the fat body or the diet. Insect hemolymph contains two lipoproteins that transport lipids, lipophorin and vitellogenin. Both are taken up via endocytosis by the oocyte, however, this provides only about 10% of the egg's lipid reserves. The rest is unloaded from circulating lipoprotein particles at the oocyte surface in the form of diacylglycerol (DAG), the major lipid transport form in insects, or as FFA. The mechanism of lipoprotein unloading at the oocyte surface is currently unclear. Possible roles of the lipid transfer particle (LTP), FA transporters, and lipoprotein lipase activity are discussed.     

Sites of Synthesis and Transport Pathways of Insect Hydrocarbons: Cuticle and Ovary as Target Tissues

SYNOPSIS. The outer surface of insects is covered with a lipidlayer that provides water-proofing and protection against environmentalstresses. Hydrocarbons (HC) are major constituents of this epicuticularwax and they also serve as semiochemicals. In some insects HCare also exploited as biosynthetic precursors for pheromones.HC are synthesized by oenocyteswhich are situated in the integumentor hemocoel. Shuttling of HC to the epicuticie, fat body, andgonads requires transport through an aqueous medium. Insects,unlike vertebrates, use a versatile lipoprotein to effect lipidtransport and to selectively deliver lipids to specific tissues.A high-density hemolymph lipoprotein (lipophorin [Lp]) servesthis function.In adult females of the German cockroach (Blattellagermanica), Lp carries both HC and a contact sex pheromone.Lipophorin is a multi-functional lipid carrier serving alsoas a juvenile hormone binding protein in many insects. Studiesofthe interactions between Lp and HC are beginning to unravelthe routes used in delivering HC to target tissues. We discussthepathways and dynamics of loading of Lp with HC and HC-derivedpheromones, their transport through the hemolymph, and depositionin various tissues, including the epicuticie, ovaries, and pheromone-emittingglands.     

Effect of phase status on responses to AKHI in the desert locust,Schistocerca gregaria gregaria

Hyperlipaemic response to adipokinetic hormone (AKH I) was demonstrated in both solitary and gregarious phases of the desert locust, Schistocerca gregaria gregaria. Time-course studies showed that the gregarious locusts had a faster response to the hormone than their solitary counterparts. At peak response time (90 min), the gregarious locusts were more sensitive to AKH I doses below 2 pmol while the solitary locusts had a higher response above this dose. Upon injection of the hormone, lipoprotein conversion occurred, resulting in the formation of the low density lipoprotein (LDLp). The LDLp formed in the gregarious locusts was much larger than that of the solitary locusts. The fat body lipid reserve (expressed as % fat body dry weight) was significantly (P < 0.01) higher in the gregarious (79.02 ± 2.77%) than in the solitary locusts (65.23 ± 2.55%). Triacylglycerol was the major lipid class representing 83.9 and 73.9% of the total lipids in gregarious and solitary locusts, respectively. The higher fat body lipid reserves and efficient LDLp formation in response to AKH in gregarious locusts compared to solitary locusts suggests a physiological adaptation for prolonged flights. © 1996 Wiley-Liss, Inc.     

A novel lipoprotein from the hemolymph of the cochineal insect, Dactylopius confusus.

A new type of insect lipoprotein was isolated from the hemolymph of the female cochineal insect Dactylopius confusus. The lipoprotein from the cochineal insect hemolymph was found to have a relative molecular mass of 450 000. It contains 48% lipid, mostly diacylglycerol, phospholipids and hydrocarbons. The protein moiety of the lipoprotein consists of two apoproteins of approximately 25 and 22 kDa, both of which are glycosylated. Both apolipoproteins are also found free in the hemolymph, unassociated with any lipid. Purified cochineal apolipoproteins can combine with Manduca sexta lipophorin, if injected together with adipokinetic hormone into M. sexta. This could indicate that the cochineal lipoprotein can function as a lipid shuttle similar to lipophorins of other insects, and that the cochineal insect apolipoproteins have an overall structure similar to insect apolipophorin-III.     

The relationships between corpora allata and fat body and haemolymph lipids in the adult female desert locust

Allatectomy of 1-day-old female desert locusts resulted in an accumulation of lipid in the fat body. This accumulation of lipid was due to the continuation of lipid deposition in the fat body after the period of somatic growth. Somatic growth and feeding activity were unaffected by allatectomy, and so could not be indirect causes of fat body lipid accumulation. Lipid accumulation in allatectomized locusts is more likely to be related directly to a lack of juvenile hormone. Implantation of active corpora allata into 1-day-old adult female locusts resulted in a premature development of oöcytes and a decrease in fat body lipid accumulation; somatic growth was not inhibited. Implantation of active corpora allata into old allatectomized locusts resulted in a decrease in the fat body lipid content and the onset of oöcyte development. The lipid synthetic activity of the fat body, measured by the incorporation of ¹⁴C-actate into total fat body lipid, was greatly increased in allatectomized locusts after the period of somatic growth. The protein synthetic activity of the fat body, measured by the incorporation of ³H-leucine into total fat body protein, remained low after the period of somatic growth in allatectomized insects. Juvenile hormone might thus have a dual effect on fat body metabolism, that is suppressing lipid synthesis and stimulating vitellogenic protein synthesis. Increased synthesis of lipid by the fat body would then account for the accumulation of lipid in the fat body after allatectomy. Inhibition of release of lipid from the fat body is unlikely to play a part in the accumulation as allatectomy had no effect on haemolymph lipid concentrations.     

Use of the glucosidase inhibitor 1,4-dideoxy-1,4-imino-D-arabinitol as a probe to investigate hypertrehalosemic hormone-mediated change in carbohydrate and lipid metabolism in the cockroach, Periplaneta americana

The glucosidase inhibitor 1,4-dideoxy-1,4-imino-D-arabinitol (DIA) was used to investigate the action of hypertrehalosemic hormone (HTH) on carbohydrate, neutral lipid, and phospholipid metabolism in the hemolymph and fat body of the cockroach, Periplaneta americana. DIA blocked the hypertrehalosemic and hyperglycemic effects of HTH, as well as the decrease in hemolymph neutral lipid and phospholipid normally induced by HTH. DIA diminished the accumulation of neutral lipid in the fat body formed under the influence of HTH and partly blocked the decrease in fat body phospholipid evoked by HTH. The increased incorporation of (14)C-glucose into fat body triacylglycerol in the presence of HTH was decreased by more than two-thirds when DIA was coinjected with the hormone. The results suggest that glucose derived from hemolymph trehalose is an important contributor to the formation of the glycerol backbone of newly formed triacylglycerol in the fat body.     

Lipid composition of the fat body and haemolymph and its relation to lipid release in Oncopeltus fasciatus

Lipid composition of the fat body and haemolymph of male milkweed bug, Oncopeltus fasciatus, was determined. Triglycerides were the predominant lipids of the fat body while diglycerides accounted for the major lipid in the haemolymph. Sterols, sterol esters, and non-esterified fatty acids were present in both fat body and haemolymph besides triglycerides and diglycerides. Only traces of monoglycerides were detected.Gas chromatographic analysis of the fatty acids revealed a difference in the fatty acid composition between fat body and haemolymph glycerides and sterol esters. Oleate and linoleate were the predominant unsaturated fatty acids in both fat body and haemolymph lipids and in the milkweed seeds as well.When fat body was labelled in vivo and in vitro with ¹⁴C-palmitate, the fatty acid was incorporated largely into the triglycerides. When the prelabelled fat body was incubated with a medium containing haemolymph the fat body released lipids mainly as diglycerides. Some radioactivity was observed in the triglycerides and non-esterified fatty acids also.Electrophoretic analysis of the incubation medium containing the haemolymph revealed that the released lipids were bound to three haemolymph lipoprotein bands. Lipid mobilization, release, and transport in Oncopeltus are discussed in relation to studies on other insects.     

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.     

Specificity and localisation of lipoprotein lipase in the flight muscles of Locusta migratoria

Using natural lipoproteins as substrates, lipase activity has been measured in leg muscle, fat body, midgut and flight muscles of Locusta migratoria. The enzymic activity in the flight muscles is higher than in those other tissues tested, confirming the potential of the flight muscles to utilise lipids at high rates. In addition, a membrane-bound lipoprotein lipase can be extracted from flight muscle. The flight muscle enzyme activity shows a marked substrate specificity; at lipoprotein concentrations equivalent to those found normally in flown or resting locusts respectively, the enzyme hydrolyses diacylglycerols associated with lipoprotein A+ (present in the haemolymph of flown or adipokinetic hormone-injected locusts) at about 4 times the rate of those associated with lipoprotein Ayellow (which is the major lipoprotein in resting locusts). In addition, the hydrolysis of lipids carried by lipoprotein Ayellow is dramatically reduced in the presence of lipoprotein A+. These observations indicate that the enzyme plays a specific role in the uptake of lipids at the flight muscles to ensure a smooth transition from carbohydrate to lipid based metabolism during flight.     

Age-related changes in the response to adipokinetic hormone in Locusta migratoria

ABSTRACT. In fifth instar nymphs of Locusta there is only a feeble adipokinetic response to extracts of corpora cardiaca. In fledglings, this poor response persists for a few days but then increases dramatically to reach a plateau by day 8. The response declines to almost zero as the locusts age beyond 35 days of adult life. This pattern of change in response is similar in both males and females but there are some differences in magnitude depending upon whether the response is measured as changes in haemolymph total lipid (vanillin-positive material) or total diglyceride (gas liquid chromatography). The poor response to adipokinetic hormone in nymphs and newly fledged locusts is not a result of shortage of stored lipid in the fat body and cannot be improved by injection of extra hormone.     

Arachnid lipoproteins: comparative aspects

Findings on hemolymph lipoproteins in the class Arachnida are reviewed in relation to their lipid and protein compositions, hydrated densities, the capacity of apoproteins to bind lipids, and the influence of xenobiotics on their structures and functionality. The occurrence of hemolymphatic lipoproteins in arachnids has been reported in species belonging to the orders Araneida, Scorpionida, Solpugida and Acarina. However, lipoproteins were properly characterized in only three species, Eurypelma californicum, Polybetes pythagoricus and Latrodectus mirabilis. Like insect and crustaceans the arachnids examined contain high density lipoproteins (HDLs) as predominant circulating lipoproteins. Although in most arachnids these particles resemble those of insect HDLs called "lipophorins", in two arachnid species they differ from lipophorins in their apoproteins, total mass and lipid composition. The hemolymph of P. pythagoricus and L. mirabilis contains another HDL of higher density, while P. pythagoricus and E. californicum hemolymph contain a third lipoprotein of very high density (VHDL). Composition of arachnid lipoproteins regarding apoprotein classes as well as lipid classes differ among species. Hemocyanin, in addition to the classical role of this protein as respiratory pigment, is presented here performing the function of apolipoprotein in some arachnid species. Reports on experiments demonstrating the capacity of hemocyanin to bind neutral and polar lipid classes, including ecdysteroids, are commented. Recent works about the changes evoked by a phosphorous pesticide on the structures and functionality of spider lipoproteins are also reviewed.     

Insect adipokinetic hormones: release and integration of flight energy metabolism

Insect flight involves mobilization, transport and utilization of endogenous energy reserves at extremely high rates. Peptide adipokinetic hormones (AKHs), synthesized and stored in neuroendocrine cells, integrate flight energy metabolism. The complex multifactorial control mechanism for AKH release in the locust includes both stimulatory and inhibitory factors. The AKHs are synthesized continuously, resulting in an accumulation of AKH-containing secretory granules. Additionally, secretory material is stored in large intracisternal granules. Although only a limited part of these large reserves appears to be readily releasable, this strategy allows the adipokinetic cells to comply with large variations in secretory demands; changes in secretory activity do not affect the rate of hormone biosynthesis. AKH-induced lipid release from fat body target cells has revealed a novel concept for lipid transport during exercise. Similar to sustained locomotion of mammals, insect flight activity is powered by oxidation of free fatty acids derived from endogenous reserves of triacylglycerol. However, the transport form of the lipid in the circulatory system is diacylglycerol (DAG) that is delivered to the flight muscles associated with lipoproteins. While DAG is loaded onto the multifunctional insect lipoprotein, high-density lipophorin (HDLp) and multiple copies of the exchangeable apolipoprotein III (apoLp-III) associate reversibly with the expanding particle. The resulting low-density lipophorin (LDLp) specifically shuttles DAG to the working muscles. Following DAG hydrolysis by a lipophorin lipase, apoLp-III dissociates from the particle, regenerating HDLp that is re-utilized for lipid uptake at the fat body cells, thus functioning as an efficient lipid shuttle mechanism. Many structural elements of the lipoprotein system of insects appear to be similar to their counterparts in mammals; however, the functioning of the insect lipoprotein in energy transport during flight activity is intriguingly different.     

昆虫卵黄原蛋白受体的研究进展

昆虫卵黄发生的一个重要过程是卵黄蛋白的摄取,已有的研究表明脂肪体合成的卵黄原蛋白(vitellogenin,Vg)是通过受体介导的内吞作用(receptor mediated endocytosis,RME)被正在发育的卵母细胞所摄取。昆虫卵黄原蛋白受体(vitellogenin receptor,VgR)是介导昆虫卵黄原蛋白胞吞作用主要受体,它属于低密度脂蛋白家族,在结构与特性上具低密度脂蛋白家族的共性。卵黄原蛋白及其受体在昆虫生殖过程中起着重要的作用,本文综述了昆虫VgR的基本特性、分子结构及表达调控等方面的研究进展。     

Hypolipemia,hypoglycemia, and inactivation of glycogen phosphorylase in Locusta migratoria

Feeding starved adult migratory locusts, Locusta migratoria, caused decreases of hemolymph lipid concentrations and of the percentage of active fat body glycogen phosphorylase which suggested that a molecule(s) from the neurosecretory system or the midgut may have been released to regulate metabolism. Fat body phosphorylase was also inactivated after insects were transferred from 0 to 25 ° C. In adults with elevated hemolymph lipid levels after the injection of small doses of corpus cardiacum extract (CC), feeding did not induce a decrease in hemolymph lipid concentrations. It appears that the processes initiated by feeding could not override the effects of the continued presence of adipokinetic hormone(s) (AKHs) in the hemolymph or their long-term effects. Aqueous, methanolic, or ethanolic extracts of brains or storage lobes (SL) of fed locust CC did not lead to decreases of hemolymph lipid concentrations. Bovine insulin was equally inactive when tested at doses which were previously reported to reduce lipid levels. Fractions of ethanolic brain extracts from 3-day-starved males collected after high-performance size-exclusion chromatography, however, produced hypoglycemic effects in fed males. Two biologically active fractions were found, one with high (≥ 10 kDa) and one with low molecular weight (approximately 1 kDa). © 1995 Wiley-Liss, Inc.     

RNA interference mediated knockdown of apolipophorin-III leads to knockdown of manganese superoxide dismutase in Hyphantria cunea

Apolipophorin-III (apoLp-III), a hemolymph protein that facilitates lipid transport in aqueous media in insects was recently shown to play a role in insect immune activation. Here, we report another novel possible function of apoLp-III in insects. To identify genes affected by apoLp-III expression in larvae, we decreased endogenous apoLp-III mRNA in Hyphantria cunea (Hc) through RNA interference; subsequently, we observed lower levels of antioxidant enzymes, including manganese superoxide dismutase (MnSOD), glutathione S-transferase, and immune proteins. Knockdown of Hc apoLp-III led to decreased MnSOD expression in fat body tissues and elevated superoxide anion levels in Hc fat body cells, suggesting that Hc apoLp-III is involved in the action and/or expression of antioxidant enzymes, especially MnSOD.     

Circulatory lipid transport: lipoprotein assembly and function from an evolutionary perspective

Circulatory transport of neutral lipids (fat) in animals relies on members of the large lipid transfer protein (LLTP) superfamily, including mammalian apolipoprotein B (apoB) and insect apolipophorin II/I (apoLp-II/I). Latter proteins, which constitute the structural basis for the assembly of various lipoproteins, acquire lipids through microsomal triglyceride transfer protein (MTP)—another LLTP family member—and bind them by means of amphipathic structures. Comparative research reveals that LLTPs have evolved from the earliest animals and additionally highlights the structural and functional adaptations in these lipid carriers. For instance, in contrast to mammalian apoB, the insect apoB homologue, apoLp-II/I, is post-translationally cleaved by a furin, resulting in their appearance of two non-exchangeable apolipoproteins in the insect low-density lipoprotein (LDL) homologue, high-density lipophorin (HDLp). An important difference between mammalian and insect lipoproteins relates to the mechanism of lipid delivery. Whereas in mammals, endocytic uptake of lipoprotein particles, mediated via members of the LDL receptor (LDLR) family, results in their degradation in lysosomes, the insect HDLp was shown to act as a reusable lipid shuttle which is capable of reloading lipid. Although the recent identification of a lipophorin receptor (LpR), a homologue of LDLR, reveals that endocytic uptake of HDLp may constitute an additional mechanism of lipid delivery, the endocytosed lipoprotein appears to be recycled in a transferrin-like manner. Binding studies indicate that the HDLp–LpR complex, in contrast to the LDL–LDLR complex, is resistant to dissociation at endosomal pH as well as by treatment with EDTA mimicking the drop in Ca²⁺ concentration in the endosome. This remarkable stability of the ligand–receptor complex may provide a crucial key to the recycling mechanism. Based on the binding and dissociation capacities of mutant and hybrid receptors, the specific binding interaction of the ligand-binding domain of the receptor with HDLp was characterized. These structural similarities and functional adaptations of the lipid transport systems operative in mammals and insects are discussed from an evolutionary perspective.