Transport and Utilization of Lipids in Insect Flight Muscles*

In migrating lepidopteran and orthopteran insects, lipid is the preferred fuel for sustained flight activity. Diacylglycerol is delivered by lipophorin to the flight muscle and hydrolyzed to free fatty acid and glycerol. After penetrating the plasma membrane by an unknown mechanism, fatty acids are bound by the intracellular fatty acid binding protein (FABP) and transported through the cytosol. After their conversion to acyl-CoA esters, the fatty acids enter the mitochondrial matrix via the carnitine shuttle for subsequent β-oxidation. This article reviews the current knowledge of lipid metabolism in insect flight muscle, with particular emphasis on the structure and function of FABP and its expression during locust development and flight.     

Fatty acid binding protein in locust and mammalian muscle. Comparison of structure,function and regulation

The flight muscle of adult desert locusts, Schistocerca gregaria, contains a fatty acid binding protein (FABP) that is homologous to mammalian M-FABP (cardiac FABP). In spite of the evolutionary distance between invertebrates and vertebrates, locust muscle FABP is similar to cardiac FABP in its amino acid sequence, structure, and binding behavior. While cardiac FABP is present already in the prenatal period, locust FABP is an adult specific protein; its expression is directly linked to metamorphosis. A correlation seems to exist between fatty acid oxidative capacity and FABP content in both locust and mammals. To accomplish the higher metabolic rate encountered during migratory flight, locust flight muscle cytosol contains more than three times as much FABP as that in mammalian heart. Increased fatty acid utilization by exercise or endurance training apparently induces FABP expression. Similarities and differences between vertebrate and invertebrate M-FABP are discussed in light of the proposed functions of muscle FABP as fatty acid transporter and cytoprotectant.     

Alternative lipid mobilization: The insect shuttle system

Lipid mobilization in long-distance flying insects has revealed a novel concept for lipid transport in the circulatory system during exercise. Similar to energy generation for sustained locomotion in mammals, the work accomplished by non-stop flight activity is powered by oxidation of free fatty acids (FFA) derived from endogenous reserves of triacylglycerol. The transport form of the lipid, however, is diacylglycerol (DAG), which is delivered to the flight muscles associated with lipoproteins. In the insect system, the multifunctional lipoprotein, high-density lipophorin (HDLp) is loaded with DAG while additionally, multiple copies of the exchangeable apolipoprotein, apoLp-III, associate with the expanding particle. As a result, lipid-enriched low-density lipophorin (LDLp) is formed. At the flight muscles, LDLp-carried DAG is hydrolyzed and FFA are imported into the muscle cells for energy generation. The depletion of DAG from LDLp results in the recovery of both HDLp and apoLp-III, which are reutilized for another cycle of DAG transport. A receptor for HDLp, identified as a novel member of the vertebrate low-density lipoprotein (LDL) receptor family, does not seem to be involved in the lipophorin shuttle mechanism operative during flight activity. In addition, endocytosis of HDLp mediated by the insect receptor does not seem to follow the classical mammalian LDL pathway.Many structural elements of the lipid mobilization system in insects are similar to those in mammals. Domain structures of apoLp-I and apoLp-II, the non-exchangeable apolipoprotein components of HDLp, are related to apoB100. ApoLp-III is a bundle of five amphipathic -helices that binds to a lipid surface very similar to the four-helix bundle of the N-terminal domain of human apoE. Despite these similarities, the functioning of the insect lipoprotein in energy transport during flight activity is intriguingly different, since the TAG-rich mammalian lipoproteins play no role as a carrier of mobilized lipids during exercise and besides, these lipoproteins are not functioning as a reusable shuttle for lipid transport. On the other hand, the deviant behavior of similar molecules in a different biological system may provide a useful alternative model for studying the molecular basis of processes related to human disorders and disease.     

Primary structure of locust flight muscle fatty acid binding protein.

The amino acid sequence of the fatty acid binding protein (FABP) from flight muscle of the locust, Schistocerca gregaria, has been determined. The sequence of the N-terminal 39 amino acid residues, determined by automated Edman degradation, was used to prepare a degenerate oligonucleotide that corresponded to amino acid residues 16-23. cDNA coding for FABP was constructed from flight muscle mRNA and amplified by the polymerase chain reaction using the degenerate oligonucleotide and an oligo dT-NotI primer adapter as primers. The amplification product was cloned and sequenced. Additionally, a cDNA library of flight muscle mRNA was prepared and screened with a 414-bp probe prepared from the clone. The primary structure of locust FABP was compared with the proteins in the Swiss protein databank and found to have significant homology with mammalian FABPs over the entire 133-residue sequence. The best match was versus human heart FABP (41% identity), attesting to the highly conserved nature of this protein. The results suggest that locust muscle FABP is a member of the lipid binding protein superfamily and may provide valuable insight into the evolution of this abundant protein class.     

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.     

Fatty-acid-binding protein from the flight muscle of Locusta migratoria: evolutionary variations in fatty acid binding

Intracellular lipid-binding proteins have evolved from a common ancestral gene with the appearance of mitochondrial oxidation, to guarantee, for example, transport of fatty acids through the aqueous cytosol to their site of utilization. The mammalian forms of these lipid carriers are structurally well-characterized and have been categorized, on the basis of sequence similarities and several typical ligand-binding features, into four subfamilies. Only a single complex structure of an invertebrate fatty-acid-binding protein (FABP) has been reported to date, which reveals a unique ligand-binding arrangement yet unknown in vertebrate FABPs. In the present study, the structure of a second invertebrate FABP (locust muscle) complexed with a fatty acid has been determined on the basis of intermolecular NOE connectivities between the protein and the uniformly (13)C-enriched oleate ligand. The resulting ligand conformation, although resembling the closely related mammalian heart- and adipocyte-type FABPs, is characterized by certain binding features that differ significantly from the typical hairpin-turn ligand shapes of the latter forms. This is primarily due to an alanine-to-leucine substitution in locust FABPs that produces a steric hindrance for ligand binding. A comparison with an FABP from tobacco hornworm larvae furthermore demonstrates that certain amino acid substitutions that appear to be specific for invertebrates decidedly influence the binding arrangement inside the protein cavity. Hence, as a result of these evolutionary variations, invertebrate FABPs may display a much greater diversity in intracellular lipid binding than observed for the mammalian transport proteins, thus possibly providing new insights for the design of modified lipid carriers.     

Fatty acid binding proteins from developing human fetal brain: Characterization and binding properties

Two fatty acid binding proteins (FABPs) of identicalM _r, 13 kDa, have been isolated from developing human fetal brain. A delipidated 105,000 g supernatant was incubated with [1 -¹⁴C]oleate and subjected to a Sephacryl S-200 column followed by gel filtration chromatography on a Sephadex G-75 column and ion-exchange chromatography using a DEAE-Sephacel column. Purity was checked by UV spectroscopy, SDS-PAGE, isoelectric focusing and immunological cross-reactivity. The two FABPs designated as DE-I (pI 5.4) and DE-II (pI 6.9) showed cross-reactivity with each other and no alteration at the antigenic site during intrauterine development. Anti-human fetal brain FABP does not cross-react with purified human fetal heart, gut, lung or liver FABPs. The molecular mass of DE-I and DE-II is lower than those of fetal lung and liver FABPs. Like liver FABP, these proteins bind organic anions, fatty acids and acyl CoAs but differ in their binding affinities. Both DE-I and DE-II have been found to exhibit higher affinity for oleate (K _d = 0.23 μM) than palmitate (K _d = 0.9μM) or palmitoyl-CoA (K _d = 0.96 μM), with DE-I binding less fatty acids than DE-II. DE-II is more efficient in transferring fatty acid from phospholipid vesjcles than DE-I indicating that human fetal brain FABPs may play a significant role in fatty acid transport in developing fetal brain.     

The recycling of protein components of the flight-specific lipophorin in Locusta migratoria

The metabolism of locust lipophorin A⁺ during lipid delivery to the flight muscle and lipid loading at the fat body was studied in vitro. Protein C₂ was shown to be released upon hydrolysis of lipophorin A⁺-carried diacylglycerol by the flight muscle lipoprotein lipase. This in vitro released protein C₂ was shown to reassociate with lipophorin A_y upon hormone-induced lipid mobilization from fat body in vitro. These results demonstrate the reversibility of the association of protein C₂ with lipophorin A_y and support the shuttle function of the protein components of locust lipophorin A⁺ in lipid transport.     

Developmental changes of FABP concentration,expression, and intracellular distribution in locust flight muscle

M-FABP from flight muscle of the locust,Schistocerca gregaria, is similar to mammalian H-FABP in its physical characteristics and amino acid sequence. We have studied developmental changes using ELISA, Northern Blotting, and EM/immuno-gold techniques. M-FABP is found in cytoplasm and nuclei, but not in mitochondria. It is the most abundant soluble muscle protein in fully developed adult locusts, comprising 18% of the total cytosolic protein. However, no FABP is detectable at the beginning of the adult stage. Its concentration rises dramatically during the next 10 days, after which it reaches its maximal value. Expression apparently is turned on after adult ecdysis and continues for 10 days; thereafter, FABP mRNA diminishes and reaches a constant, but low level, probably needed to maintain the current FABP level. From a series of experiments employing metamorphosis-controlling hormones and antihormones it is evident that the induction of FABP expression is directly linked to metamorphosis.Abbreviations ELISA Enzyme Linked Immuno Sorbent Assay - FABP Fatty Acid-Binding Protein - H-FABP mammalian Heart Fatty Acid-Binding Protein - M-FABP locust flight Muscle Fatty Acid-Binding Protein     

Cytoplasmic fatty acid-binding proteins: emerging roles in metabolism and atherosclerosis

Cytoplasmic fatty acid-binding proteins (FABPs) are a family of proteins, expressed in a tissue-specific manner, that bind fatty acid ligands and are involved in shuttling fatty acids to cellular compartments, modulating intracellular lipid metabolism, and regulating gene expression. Several members of the FABP family have been shown to have important roles in regulating metabolism and have links to the development of insulin resistance and the metabolic syndrome. Recent studies demonstrate a role for intestinal FABP in the control of dietary fatty acid absorption and chylomicron secretion. Heart FABP is essential for normal myocardial fatty acid oxidation and modulates fatty acid uptake in skeletal muscle. Liver FABP is directly involved in fatty acid ligand signaling to the nucleus and interacts with peroxisome proliferator-activated receptors in hepatocytes. The adipocyte FABP (aP2) has been shown to affect insulin sensitivity, lipid metabolism and lipolysis, and has recently been shown to play an important role in atherosclerosis. Interestingly, expression of aP2 by the macrophage promotes atherogenesis, thus providing a link between insulin resistance, intracellular fatty acid disposition, and foam cell formation. The FABPs are promising targets for the treatment of dyslipidemia, insulin resistance, and atherosclerosis in humans.     

The activities of lipases and carnitine palmitoyl-transferase in muscles from vertebrates and invertebrates

1. The activities of tri-, di- and mono-glyceride lipase and carnitine palmitoyltransferase were measured in homogenates of a variety of muscles. These activities were used to estimate the rate of utilization of glycerides and fatty acids by muscle. In muscles whose estimated rates of fat utilization can be compared with rates calculated for the intact muscle from such information as O₂ uptake, there is reasonable agreement between the estimated and calculated rates. 2. In all muscles investigated the maximum rates of hydrolysis of glycerides increase in the order triglyceride, diglyceride, monoglyceride. The activity of diglyceride lipase is highest in the flight muscles of insects such as the locust, waterbug and some moths and is lowest in the flight muscles of flies, bees and the wasp. These results are consistent with the utilization of diglyceride as a fuel for some insect flight muscles. 3. In many muscles from both vertebrates and invertebrates the activity of glycerol kinase is similar to that of lipase. It is concluded that in these muscles the metabolic role of glycerol kinase is the removal of glycerol produced during lipolysis. However, in some insect flight muscles the activity of glycerol kinase is much greater than that of lipase, which suggests a different role for glycerol kinase in these muscles.     

Lipid storage and changes during flight by triatomine bugs (Rhodnius prolixus and Triatoma infestans)

Exceptionally large amounts of lipid are stored in flight muscles of Rhodnius prolixus and Triatoma infestans (197 and 90 μmoles glyceride glycerol per g fresh weight respectively). The bulk of this lipid is in the form of triacylglycerol.A significant decrease in the muscle lipid occurs during the first hour of flight. Over the same period there is an increase in haemolymph lipid (particularly of diacylglycerol) which is taken to indicate the use of lipid from the fat body. The carbohydrate content of muscle and haemolymph is low, so it is likely that the supply of energy for flight is provided almost exclusively by the oxidation of fat. Oleate and palmitate are the major fatty acid components of lipid from both Triatoma and Rhodnius and are probably also the major fatty acids used for oxidation.Maturation of flight ability is temporally associated with the development of flight muscle size and increase in glyceride content.     

Fatty acid oxidation by the flight muscles of the dragonfly, Pantala flavescens

The dragonfly, Pantala flavescens, remains air-borne for many hours and should be expected to utilize fat during its prolonged flight. In vitro studies on fatty acid oxidation in the flight muscles of this insect have revealed that the muscles are capable of oxidizing butyrate, octanoate, palmitate, and stearate. However, there seems to be a preferential oxidation of short chain fatty acids. Added carnitine appears to have a stimulatory effect on palmitic acid oxidation in the homogenate as well as in the mitochondrial preparation.     

The fatty acid-binding protein from human skeletal muscle

Fatty acid-binding protein (FABP) was isolated from human skeletal muscle by gel filtration and anion- and cation-exchange chromatography. The isolation procedure, however, with rat and pig skeletal muscle gave mostly inactive preparations. Rat muscle FABP preparations contained parvalbumin as a contaminant. FABP from human muscle had a Mr of about 15 kDa, a pI value of 5.2, and a Kd value with oleic acid of 0.50 microM. Skeletal muscle and heart FABPs and their antisera showed a strong cross-reactivity on Western blots and in enzyme-linked immunosorbent assays (ELISA). No cross-reactivity was observed with liver FABP and its antiserum. On the basis of amino acid composition, electrophoretic behavior, fatty acid binding, and immunochemical properties, human skeletal muscle FABP must be similar or closely related to human heart FABP. The FABP content determined by ELISA was comparable in various human muscles and cultured muscle cells, but lower than that in rat muscles.     

Characteristics of fatty acid-binding proteins and their relation to mammary-derived growth inhibitor

Summary Based on sequence relationships the cytoplasmic fatty acid-binding proteins (FABPs) of mammalian origin are divided into at least three distinct types, namely the hepatic-, intestinal- and cardiac-type. Highly conserved sequences of FABPs within the same type correlate with immunological crossreactivities. Isoforms of hepatic-type FABP are found in several mammalian species and for bovine liver FABP specific shifts in isoelectric points upon lipidation with fatty acids are observed. Isoforms of intestinal-type FABP are not known and the occurrence of cardiac-type isoforms so far is confined to bovine heart tissue. A bovine mammary-derived growth inhibitor (MDGI) is 95% homologous to the cardiac-type FABP from bovine heart. Dissociation constants of FABP/fatty acid complexes are in the range of 1 M and 1:1 stoichiometries are usually found, but the neutral isoform of hepatic FABP from bovine liver binds 2 fatty acids. On subcellular levels hepatic- and cardiac-type FABPs are differently distributed. Though mainly cytosolic in either case, immunoelectron microscopy as well as a gelchromatographic immunofluorescence assay demonstrate the association of hepatic FABP in liver cells with microsomal and outer mitochondrial membranes and with nuclei, whereas in heart cells cardiac FABP is confined to mitochondria' matrix and nuclei. In mammary epithelial cells MDGI is associated with neither mitochondria nor endoplasmic reticulum, and is expressed in a strictly developmental-dependent spatial and temporal pattern. The specific role proposed for MDGI is to arrest growth of mammary epithelial cells when they become committed to differentiation in the mammary gland.     

Fatty acid-binding protein and its relation to fatty acid oxidation

A relation between fatty acid oxidation capacity and cytosolic FABP content was found in heart and various muscles of the rat. Other tissues do not show such a relation, since they are involved in more or other pathways of fatty acid metabolism. At postnatal development FABP content and fatty acid oxidation capacity rise concomitantly in heart and quadriceps muscle in contrast to in liver and kidney. A dietary fat content of 40 en. % increased only the FABP content of liver and adipose tissue. Peroxisomal proliferators increased fatty acid oxidation in both liver and kidney, but only the FABP content of liver, and had no effect on heart and skeletal muscle. The FABP content of muscle did not show adaptation to various conditions. Only it increased in fast-twitch muscles upon chronic electrostimulation and endurance training.