Michael A. Wells

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.

Lipophorin: The Structure of an Insect Lipoprotein and Its Role in Lipid Transport in Insects

Publisher Summary A significant way of organizing a lipid transport system is provided by insect lipoproteins, which provide an interesting contrast to the mammalian lipid transport system. In insects, a single lipoprotein, lipophorin, acts as a reusable shuttle to transfer diglycerides between the fat bodies, where they are stored as triglycerides, and the peripheral tissues. This chapter describes the composition and the organization of lipids and proteins in the lipophorins and the structure of apolipophorin-III that is determined by X-ray crystallography to 2.5 A. Lipophorin is reloaded with lipid by another remarkable protein, the lipid transfer particle, which can affect a net transfer of diglycerides from the fat bodies to the depleted lipophorin particles. The major progress that is achieved in describing the lipid transport system in insects provides the results of significance for understanding lipid transport in general, an illustration of comparative biochemistry at its best. The only common major lipid component of vertebrate and insect lipoproteins is phospholipid (PL). The physiological roles of lipophorin in the biosynthesis and metabolism are also discussed.

Early Trypsin Activity is Part of the Signal Transduction System that Activates Transcription of the Late Trypsin Gene in the Midgut of the Mosquito, Aedes aegypti

Trypsin activity during the first hours after feeding is essential to induce late trypsin gene expression. These results are consistent with the idea that free amino acids or other products released during digestion might be the initial signal for transcriptional activation of late trypsin. Besides early trypsin, some other factor(s) have to be translated for induction of late trypsin. This is the first case in which the proteolytic activity of a digestive enzyme is part of the signal transduction system which regulates expression of a second gene. The presence of two trypsins allows the mosquito to assess the quality of the meal and adjust the levels of late trypsin for a particular meal with remarkable flexibility.

Sequence of three cDNAs encoding an alkaline midgut trypsin from manduca sexta

We have purified trypsin from the midgut of Manduca sexta and shown it has an alkaline pH optimum of 10.5. In order to clone the midgut trypsin, a DNA probe was generated using the polymerase chain reaction (PCR) with template isolated from a midgut cDNA library phage stock, a mixture of degenerate primers synthesized to code for the highly conserved region around the active site serine found in trypsins, and the T7 sequencing primer. Three different trypsin cDNAs were isolated each of which encodes a preproenzyme of 256 amino acids with a putative signal sequence of 17 amino acids, an activation peptide of seven amino acids and a mature trypsin of 232 amino acids. The encoded midgut trypsins contain the highly conserved residues, Asp, His, Ser, involved in catalysis in serine proteases, along with the residues which define the trypsin specificity pocket. Sequence comparisons show that all sequences are similar to other invertebrate and vertebrate serine proteases, but they differ in that two of the three encoded trypsins have an odd number of cysteines. Northern analysis localizes the trypsin mRNA to the middle third of the midgut. A large number of arginines (19, 20 and 21) are encoded by the three cDNAs which may stabilize the trypsin, by remaining protonated, in the alkaline midgut of M. sexta.

cDNA and deduced amino acid sequence of a blood meal-induced trypsin from the mosquito, Aedes aegypti

A cDNA for a midgut trypsin induced by a blood meal has been cloned and sequenced from the mosquito Aedes aegypti. The 862 base sequence codes for a 257 amino acid protein, which is presumably a trypsin precursor, since the sequence of purified mosquito trypsin begins at residue 26, immediately following an arginine residue in the precursor. The amino terminal 25 amino acids in the precursor are composed of a putative 15 amino acid signal peptide and a 10 amino acid activation peptide. Compared to vertebrate trypsinogens, the putative mosquito trysinogen lacks the four acidic amino acids immediately preceding the basic amino acid at the activation peptide cleavage site, suggesting the mechanism of its activation may be different from the activation of vertebrate trypsinogens. However, since a trypsinogen has not yet been isolated from the mosquito, these conclusions are tentative. The deduced amino acid sequence is homologous to that of other trypsins in those residues around the catalytic triad, and in several residues which are found only in trypsins. However, the sequence of the specificity pocket in mosquito trypsin, KESPC, differs from that found in other trypins, KDSC. The Asp is thought to bind the basic residue of the substrate, and the Glu in the mosquito trypsin may serve the same role. The changes in trypsin protein and mRNA levels following a blood meal indicate that an important component of the regulation of trypsin synthesis is at the transcriptional level.

Digestion, absorption, transport and storage of fat during the last larval stadium of Manduca sexta. Changes in the role of lipophorin in the delivery of dietary lipid to the fat body

Abstract Using radiolabeled triolein, it was shown that in Manduca sexta larvae dietary triacylglycerol was hydrolyzed completely to free fatty acid in the midgut before absorption into midgut cells. Subsequently, the fatty acids enter the midgut cell where they are found in phospholipid, di- and triacylglycerol and leave the midgut as diacylglycerol and are transported by lipophorin. In feeding larvae, lipophorin diacylglycerol was rapidly cleared from the hemolymph and most of the label was found in fat body triacylglycerol. In contrast, diacylglycerol was cleared very slowly from wanderer hemolymph and only a very small amount was taken up by the fat body. In vitro studies showed that these differences in uptake of diacylglycerol from lipophorin were a result of alterations in properties of fat body during development. Further in vitro studies showed that when lipophorin was incubated with feeding stage fat body more than 95% of the diacylglycerol was taken up by the fat body but none of the lipophorin protein entered the fat body.

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.

Early trypsin, a female-specific midgut protease in Aedes aegypti: Isolation, amino-terminal sequence determination, and cloning and sequencing of the gene

Early trypsin is a female-specific protease present in the Aedes aegypti midgut during the first hours after ingestion of a blood meal. It plays an essential role in the transcriptional activation of the late trypsin form, the major midgut endoprotease involved in the blood meal digestion. Early trypsin is the most abundant midgut polypeptide isolated by benzamidine-sepharose affinity chromatography 3 h after feeding. The amino-terminal sequence of the early trypsin protein matches that of the 3a1 cDNA for a putative trypsinogen described by Kalhok et al. (Insect. Molec. Biol., 2, 71-79, 1993). The early trypsin cDNA was over expressed in Escherichia coli. Polyclonal antibodies generated against this recombinant protein were used to show that the enzyme was present in the midgut during the first 4 h after feeding. A 2.5 kb genomic clone of the early trypsin was isolated, mapped and subcloned. A 1.56 kb subclone, corresponding to 1303 bp of the upstream regulatory region and 265 bp of the coding region, was sequenced. The gene contains a 64 nucleotide intron which interrupts the codon for Val at position 18 of the protein. This Val is located toward the end of the putative signal sequence of the protein.

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.

cDNA cloning and pattern of expression of an adult, female-specific chymotrypsin from Aedes aegypti midgut

A cDNA for a midgut chymotrypsin, induced by a blood meal, has been cloned and sequenced from the mosquito Aedes aegypti. The 938 base sequence codes for a 268 amino acid protein, which contains an 18-residue signal peptide and a seven-residue activation peptide. The deduced amino acid sequence contains several features typical of chymotrypsin proteases, including the catalytic triad of serine proteases and the residues that determine the chymotrypsin substrate specificity pocket. The chymotrypsin mRNA, absent in larvae, pupae, males and newly emerged females, reaches detectable levels within 24 h post-emergence and attains a maximum level 3-7 days after emergence. Translation of the chymotrypsin mRNA is induced by feeding a protein meal, and there is a dramatic increase in midgut chymotrypsin enzymatic activity after feeding. Chymotrypsin activity remained high during protein digestion, but chymotrypsin protein levels and enzymatic activity were almost undetectable once digestion was completed, 48 h after feeding.