Hemolymph Lipoprotein Research Articles

Isolation and partial characterization of the haemolymph lipoproteins of the wax moth, Galleria mellonella

Three lipoproteins differing in hydrated density were isolated from the larval haemolymph of the wax moth, Galleria mellonella by preparative ultracentrifugal floatation procedures. They are the two high density lipoproteins (LP-Ia; d = 1.105–1.10 and LP-Ib; d = 1.152–1.160) and the very high density lipoprotein (LP-II; d=1.265). The total lipid content in LP-Ia amounts to 42% by weight, in LP-Ib it amounts to 22% and in LP-II to approximately 6%. All three lipoproteins contained a carbohydrate moiety in addition to lipid and protein moieties. About 70% of the lipid in the haemolymph is bound to LP-Ia and in all three lipoproteins, diacylglycerol is the predominant lipid, but appreciable amounts of phospholipids and free fatty acids are also present. Phosphatidylethanolamine and phosphatidylcholine are the predominant phospholipids present in the lipoproteins. The wax moth lipoproteins in general resemble the lipoproteins of the American silkmoths in lipid and protein content, lipid composition and in ultracentrifugal and electrophoretic properties.

Lipid absorption by isolated intestinal preparations

Locusts were fed [1- 14C]-trioleylglycerol alone or mixed with trioleyl-[ 3H]-glycerol. Thirty minutes after ingestion the intestine was excised and incubated in dilute haemolymph. Lipolysis continued under these conditions. Glycerol was rapidly transported to the haemolymph. The fatty acids were taken up by the mucosa, incorporated into intestinal wall phospholipids, di- and tri-acylglycerols. Considerable amounts of labelled diacylglycerol were released into the haemolymph; fatty acids were also detected in the haemolymph. The 3 H 14 C ratio of the intestinal phospholipids and diacyl-glycerol was very similar to that of the haemolymph diacylglycerol. When [ 3H]-labelled palmitate and [ 14C]-glycerol dissolved in buffered saline were pumped through isolated intestinal preparations almost identical results were obtained. Glycerol was rapidly absorbed and palmitate was incorporated into intestinal wall phospholipids and neutral lipids. Both labelled diacylglycerol and fatty acids were detected in the haemolymph lipoproteins.

The juvenile hormone-carrier in the haemolymph of the acridine grasshopperGomphocerus rufus L.: Blocking of the juvenile hormone's action by means of repeated injections of an antibody to the carrier.

Egg-production, spermathecal enzyme activity and sexual behaviour inGomphocerus rufus females are shown to be controlled by the corpora allata, i.e. the juvenile hormone (JH). The haemolymph of this insect contains a high molecular weight glyco-lipo-protein (220,000; IEP 6.8) which has a high affinity for JH (Kd=5×10-8M). This protein binds JH selectively, even in competition with other haemolymph lipoproteins. Purification of the JH-binding protein and subsequent immunisation of rabbits yielded a homogenous antibody solution (Anti-JCv) which eliminated the JH-transport in the haemolymph if it was injected into adult males or females for several days after ecdysis. After this treatment the females did not produce eggs, and spermatophore-formation occurred only partially in the males, this indicating that the Anti-JCv-injections had exactly the same effect as bilateral allatectomy. The haemolymph of related grasshoppers was shown to contain a protein which is immunologically identical to the JH-carrier from theG. rufus haemolymph. It is suggested that the JH produced by the c. allata needs a protein carrier in order to exert its control on the functioning of the reproductive system. The synthesis of the JH-carrier is not dependent upon the presence of JH, i.e. the c. allata, since it is also produced by bilaterally allatectomized males and females throughout their whole life.

Adipokinetic hormone and the regulation of carbohydrate and lipid metabolism in a working flight muscle preparation

Trehalose utilisation in a working flight muscle preparation is reduced, in a competitive manner, in the presence of haemolymph lipoprotein. In the presence of trehalose, utilisation of lipid from lipoprotein preparations with high lipid: protein ratios (but not low lipid: protein ratios) is favoured when a tissue extract containing adipokinetic hormone is included in the incubation medium. Under these conditions trehalose utilisation is reduced to a minimum level. This inhibition of trehalose oxidation in the presence of high-lipid lipoprotein and adipokinetic hormone is completely removed by the addition of 2-bromostearic acid to the incubation medium. Glycerol utilisation is not inhibited when lipid oxidation is favoured by the addition of adipokinetic hormone. It is argued that glycolytic flux is reduced (when lipid oxidation is favoured) at a point before the formation of triose phosphates; perhaps at the aldolase reaction.

Further characterization of lepidopteran vitellogenin from haemolymph and mature eggs

Vitellogenin was isolated and purified from the pupal haemolymph and the eggs of Cynthia silkworm by the method originally developed for purifying the haemolymph lipoprotein II. The purified vitellogenin was characterized according to substructure, sugar composition, and lipid composition. The vitellogenin molecule consists of two large subunits and two small subunits, and the estimated mol. wt are 230,000 and 55,000, respectively. Mannose is present in vitellogenin, and, based upon the protein amount, its content is approximately 2.5% for both haemolymph-vitellogenin and egg-vitellogenin. Both egg-vitellogenin and haemolymph-vitellogenin contain diacylglycerol, cholesterol, and phospholipids, but the concentration of diacylglycerol in egg-vitellogenin is lower than that in haemolymph-vitellogenin. From these data, a mechanism of uptake of vitellogenin into oöcytes is discussed.

Isolation and characterization of insect vitellogenin. its identity with hemolymph lipoprotein II

Several years ago, we isolated two major lipoproteins from the pupal hemolymph of the Cynthia silkworm, both of which contained diacylglycerol as a major lipid, and named them lipoproteins I and II. In this paper, we present definite evidence that the vitellogenin (female protein) of this insect is indeed identical with lipoprotein II.

Exogenous lipid transport by the haemolymph lipoproteins in Periplanet a americana

To investigate the transport of exogenous lipids by the haemolymph lipoproteins, two kinds of experiments were carried out: in some experiments the insects were given a test-meal of double labelled tripalmitin (U 3H glycerol, 1- 14C palmitic acid) and were killed either 2 hr or 6 days later. In other experiments each insect received 2 identical test-meals: (9–10 3H palmitic acid) tripalmitin and 6 days later (1- 14C palmitic acid) tripalmitin. The insects were killed 2 hr after the ingestion of the second test meal. The lipids bound to the lipoproteins from the different fractions separated by electrophoresis of the haemolymph were analysed. The amount of labelled lipids transported by the haemolymph was much higher at 2 hr than at 6 days. The ingested triglycerides were absorbed unchanged or resynthetized from the products of their hydrolysis. The lipids released from the midgut into the haemolymph were found to be associated to lipoproteins carrying high amounts of triglycerides and exhibiting little or no electrophoretic mobility; the lipids released from the fat body into the haemolymph were mostly diglycerides; the glycerol released from the hydrolysis of exogenous triglycerides was not used in resynthesis of glycerides.

Effect of dietary cholesterol deficiency upon its distribution in the larvae of the housefly, Musca domestica

The free sterol, total phospholipids and protein content of the various tissues and haemolymph lipoproteins obtained from the larvae of Musca domestica, reared on the diets containing 0.56 μmole cholesterol/g wet weight of diet (normal) and 0.05 μmole cholesterol/g wet weight of diet (deficient) have been determined. The cholesterol in the diet was found to be taken up by the larvae and distributed between all the tissues examined. About 60% of the free sterol in the larvae was recovered from the composite gut fraction and muscle. Cholesterol deficiency reduced both the growth of larvae and the free sterol content of the various tissues and haemolymph when compared to that of normal larvae. Cholesterol deficiency resulted in a slightly higher proportion of sterol and protein of the larval haemolymph being associated with the lipoproteins having slower electrophoretic mobility. Most of the different tissues from the cholesterol deficient larvae contained a much smaller proportion of their normal free sterol content than of their phospholipid or protein; the brain tissue however contained a higher percentage of free sterol and the haemolymph a much lower percentage than would be expected from the lowering of phospholipid and protein content as a result of the deficiency. When the sterol content was expressed relative to the protein, the ratio was higher in the brain tissue of both the normal and deficient larvae than the ratio present in the remaining tissues, apart from the composite gut fraction of the normal larvae. The results suggest that a disproportionate amount of available cholesterol was being concentrated into the nervous system of the cholesterol deficient insect. A rather higher proportion of the total sterol fraction recovered from the various tissues and haemolymph lipoproteins of cholesterol deficient larvae behaved as ‘polar metabolites’ of cholesterol when compared with that of normal larvae.

Role of the "thrombocytoids" in capsule formation in the dipteran Calliphora erythrocephala.

Of the three hemocyte types present in the blood of Calliphora, only one participates in capsule formation around implanted cellophane. This hemocyte, the thrombocytoid, shows in the blood a tendency to dissociate into numerous small cytoplasmic fragments, comparable to the mammalian megakaryocyte. This tendency is dramatically increased during the process of encapsulation. Most of the intact thrombocytoids and the numerous fragments participating in capsule formation do not show any particular modifications in their cytoplasm during this process, which corresponds to a mere sequestration of the implant. Dense material, resulting from necrotic cell debris and hemolymph lipoproteins, is often observed between the cellophane and encapsulating thrombocytoids, which apparently participate in the resorption of this material.

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.

Haemolymph proteins and lipoproteins in lepidoptera—A comparative electrophoretic study

1. 1. Polyacrylamide gel electrophoresis of haemolymph from seven species of Lepidoptera ( Hyalophora cecropia, H. gloveri, Callosamia promethea, Philosamia cynthia, Antheraea polyphemus, A. mylitta, Manduca sexta) reflected the close relationship between species within the same genus and the differences between the saturniids and a sphingid. 2. 2. Comparisons were made of ontogenetic protein pattern changes in the larval-pupal-adult development of P. cynthia (a saturniid) and M. sexta (a sphingid). 3. 3. With the exception of M. sexta, it was possible to recognize the sex of pupae and adults from the electropherograms by the presence of a major protein in the haemolymph of pupal and adult females which is absent from or present in lower concentration in the males. In all seven species, the total protein concentration of the haemolymph is higher in females than in males. 4. 4. All species examined have haemolymph lipoproteins, and all the haemolymph lipoproteins of H. cecropia, H. gloveri, P. cynthia, C. promethea and A. polyphemus are also glycoproteins. Several proteins and lipoproteins possessing esterase activity were detected in the larval or pupal haemolymph of M. sexta, H. gloveri, H. cecropia, P. cynthia and A. mylitta.

Studies on phospholipid transport by haemolymph lipoproteins

Pupae of the silkmoth, Philosamia cynthia, were injected with [ 32P]orthophosphate. The two major haemolymph lipoproteins, lipoproteins I and II, were purified and the radioactivity incorporated into phospholipids associated with the lipoproteins was assayed. It was found that almost all of the [ 32P]phospholipids in the haemolymph were bound to the two lipoproteins. Transfer of [ 32P]phospholipids between lipoproteins I and II was also demonstrated in vitro. The in vitro experiment of phospholipid release from fat body into haemolymph was carried out and resulted in failure, because of limitation of incubation time. Since haemolymph itself is not capable of synthesizing phospholipids, however, it is very probable that phospholipids associated with lipoproteins I and II are not only constituents of the lipoproteins, but also transported by these lipoproteins.

Triglyceride digestion and absorption in the locust, locust a migratoria

Locusts were fed glyceryl tri[I −14C]oleate. The triglyceride was rapidly hydrolyzed and 14C-labeled fatty acids were found in the lumen. Large amounts of 14C-labeled acids were found in the phospholipids of the intestinal wall, hemolymph diglycerides and fat body glycerides. Only trace amounts of free acids were detected in these tissues. When double labeled triolein, [2 −3H]glyceryl tri[I −14C]oleate was fed, the 3H/ 14C ratio of the lipids isolated from the above tissues was very markedly lower than that of the fed triolein. Free [ 3H]glycerol was found. To test whether fatty acids or diglycerides were released from the intestine into the hemolymph, the following observations were made: (I) when bovine serum albumin was injected into the hemocoel soon after feeding, 14C-labeled fatty acids could be isolated from the hemolymph. (2) [ 14C]Oleic acid was incorporated into hemolymph lipoproteins. The acid was rapidly utilized in vitro by the fat body and subsequently 14C-labeled diglycerides were found in the lipoproteins. (3) Diglyceride uptake in vitro by the fat body was demonstrated.

The effect of dietary changes on the phospholipid composition of the haemolymph lipoproteins of larvae of the housefly, Musca domestica

Qualitative and quantitative analyses have been made of the phospholipid composition of haemolymph from larvae of Musca domestica and of the two lipoprotein fractions separated from it by agarose-gel electrophoresis. The effect of rearing the larvae on defined diets containing adequate choline, no added choline, and choline plus 2-aminobutan-1-ol on this composition has been studied. The haemolymph lipoproteins have a phospholipid pattern similar to that of the unfractionated haemolymph. The chief component is phosphatidylethanolamine. Diglycerides and free sterols are the major neutral lipids present in the haemolymph and the separated lipoproteins. The different diets cause changes in the various phospholipids present in the two lipoproteins which are similar to those that occur in other tissues of the larvae. Choline deficiency increased the proportion of the haemolymph phospholipid that is associated with the lipoprotein having the slower electrophoretic mobility. The results are compared with those obtained from other insects and vertebrates and the rôle of the lipoprotein in choline deficiency in the housefly is discussed.

Complement-Unlike Hemolytic Activity in Lobster Hemolymph

Abstract Hemagglutinating and cobra venom factor (CVF)-activated hemolytic activities were found in the hemolymph of the lobster, Homarus americanus. The substances in hemolymph responsible for these two activities were separated and shown to be mutually independent. Hemolytic activity was due to the action of a low level of phospholipase A in the CVF preparation on lobster hemolymph lipoprotein. Lecithin was converted to lysolecithin. Phospholipase A activity was also demonstrated in a commercial preparation of CVF. Lecithin and human lipoproteins as well as lobster lipoproteins were suitable substrates for both CVF preparations.

Haemolymph lipoprotein transport of juvenile hormone

Studies utilizing Sephadex chromatography, slab gel electrophoresis, thin-layer chromatography, and bioassay indicate that the yellow high-density lipoprotein (yHDL) found in the haemolymph of saturniid moths is involved in the transport of juvenile hormone. Although all six haemolymph lipoproteins can bind 14C-2-juvenile hormone in vitro, only the yHDL and yellow very highdensity lipoprotein (yVHDL) bind juvenile hormone in vivo. Pupal yHDL binds exogenously applied juvenile hormone even though this developmental stage normally does not have juvenile hormone. When yHDL from a stage (male adult) with a high juvenile titre is bioassayed, the results were positive whereas normal pupal yHDL and adult male haemolymph proteins minus the yHDL were ineffective. Further studies revealed that 14C-2-juvenile hormone is converted to a more polar compound(s) both in haemolymph in vitro and when injected into pharate adults. This material is accumulated by the gut and may be an excretory product. Material less polar than juvenile hormone was detected by thin-layer chromatography. Preliminary isotope dilution experiments suggest that 1 mg yHDL is capable of binding a minimum of 60 μg of juvenile hormone although this large amount is never present in the insect.

Studies on Hormone Recognition by Arthropod Target Tissues

Numerous experiments have implicated the crustacean hepatopancreas as a target of the molting hormone, and recent studies strongly suggest that β-ecdysone is the crustacean molting hormone. For example β-ecdysone can elicit molting in niter-molt crustaceans and stimulates the synthesis of protein and RNA in the hepatopancereas. When 3H-α-ecdysone is injected into premolt crayfish, it is efficiently excreted, but label can be detected in almost all tissues examined. To determine whether target tissues contain specific “receptor” molecules which distinguish those tissues that respond to molting hormone from those that do not, a series of in vivo and in vitro experiments were conducted utilizing Sephadex chtomatography and sucrose density gradient ultracentrifugation. These studies reveal the presence of two proteins in the cytosol fraction of the hepatopancreas that bind label from the 3H-α-ecdysone. These proteins have approximate molecular weights of 250,000 (11.3S) and 130,000 (6.35S), and the larger may be an aggregate of the smaller. Micro-chemical analysis demonstrated that the label associated with these proteins is no longer attributable to α-ecdysone nor any ecdysone-like compound thus far described. Two alternate structures are described that may represent the metabolite bound to the protein, and it is suggested that the binding protein-metabolite complex may be the the active from of the molting hormone. Studies are also described dealing witha hemolymph lipoprotein in silkmoths that binds juvenile hormone and it is postulated that the water soluble hemolymph lipoprotein-juvenile hormone complex is the means by which the lipoidal juvenile hormone is transported in the aqueous hemolymph.

Studies on the synthesis of lipoproteins during larval-pupal development of Hyalophora cecropia

The synthesis of haemolymph lipoproteins at 3 developmental stages during larval-pupal development of the silkworm, Hyalophora cecropia, has been studied. The haemolymph lipoprotein concentration is very low in the fourth-instar larva, but increases sharply in the fifth-instar larva and reaches a maximum in the pharate pupa. Fat body from fifth-instar larvae, pharate pupae, and newly ecdysed pupae incorporated labelled lipid and protein precursors into lipoproteins, and when such fat body was incubated with haemolymph labelled lipoproteins were recovered from the haemolymph. Puromycin (2 mg. per ml.), when added to the incubation medium, partially inhibited the incorporation of labelled amino-acids into the protein moiety of the lipoproteins, but the incorporation of labelled fatty acids into the lipid moiety was unaffected. Fat body extracts (from which the lipids had been removed) incorporated labelled haemolymph lipids into lipoproteins when incubated in vitro. Radioassay of the lipid fractions from these lipoproteins assembled in vitro revealed a similarity to those of the native haemolymph lipoproteins after incubation with prelabelled fat body. From the data it is suggested that the fat body may contain apo-lipoproteins that are able to bind with haemolymph lipids to form lipoproteins.

The uptake and transport of cholesterol by haemolymph lipoproteins

Larvae of the silkworm, Philosamia cynthia, were fed on an artificial diet containing [ 14C]cholesterol. After pupation the two major haemolymph lipoproteins, lipoproteins I and II, were purified and radioassayed. Almost all of the [ 14C]cholesterol found in the haemolymph was associated with the two lipoproteins. The quantity of [ 14C]cholesterol associated with lipoprotein I was much greater than that associated with lipoprotein II per unit weight of protein. In vitro experiments were conducted in which purified lipoproteins I and II were incubated with isolated midgut preparations from larvae fed [ 14C]cholesterol. It was demonstrated that lipoprotein I specifically takes up cholesterol from the midgut but that lipoprotein II does not appear to have this capability. [ 14C]Cholesterol can be transferred between lipoproteins I and II in vitro. There was no significant incorporation of [ 14C]cholesterol into either lipoprotein I or II when haemolymph was placed in contact with [ 14C]cholesterol in vitro, suggesting that the midgut is required for sterol uptake by these insect haemolymph lipoproteins.

Studies on ecdysone transport in insect haemolymph

Philosamia cynthia larvae and pupae were fed or injected with high specific activity 3H-α-ecdysone. The two major haemolymph lipoproteins were purified and the 3H-ecdysone was radioassayed during the purification process. The data reveal that α-ecdysone is not bound to these lipoproteins. When the total haemolymph protein content was separated by gel filtration, there was no overlap between the protein and 3H-ecdysone fractions. These experiments suggest that α-ecdysone is transported in the haemolymph as ‘free’ hormone although the possibility remains that the techniques utilized resulted in the breaking of existing bonds.