Lipid transfer particle mediates the delivery of diacylglycerol from lipophorin to fat body in larval Manduca sexta

This work analyzed the process of lipid storage in fat body of larval Manduca sexta, focusing on the role of lipid transfer particle (LTP). Incubation of fat bodies with [(3)H]diacylglycerol-labeled lipophorin resulted in a significant accumulation of diacylglycerol (DAG) and triacylglycerol (TAG) in the tissue. Transfer of DAG to fat body and its storage as TAG was significantly inhibited (60%) by preincubating the tissue with anti-LTP antibody. Lipid transfer was restored to control values by adding LTP to fat body. Incubation of fat body with dual-labeled DAG lipophorin or its treatment with ammonium chloride showed that neither a membrane-bound lipoprotein lipase nor lipophorin endocytosis is a relevant pathway to transfer or to storage lipids into fat body, respectively. Treatment of fat body with suramin caused a 50% inhibition in [(3)H]DAG transfer from lipophorin. Treatment of [(3)H]DAG-labeled fat body with lipase significantly reduced the amount of [(3)H]DAG associated with the tissue, suggesting that the lipid is still on the external surface of the membrane. Whether this lipid represents irreversibly adsorbed lipophorin or a DAG lipase-sensitive pool is unknown. Nevertheless, these results indicate that the main pathway for DAG transfer from lipophorin to fat body is via LTP and receptor-mediated processes.     

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.     

Insect lipid transfer particle can facilitate net vectorial lipid transfer via a carrier-mediated mechanism.

The mechanism of facilitated lipid transfer by insect or mammalian plasma lipid transfer proteins has not been elucidated. Transfer catalysts may act as carriers of lipid between donor and acceptor lipoproteins or, alternatively, transfer may require formation of a ternary complex. This study was designed to determine if Manduca sexta hemolymph lipid transfer particle (LTP) can facilitate net vectorial transfer of lipid without concomitant contact between donor and acceptor lipoproteins and LTP. M. sexta [3H]diacylglycerol-high density lipophorin-larval ([3H]DAG-HDLp-L) and human low density lipoprotein (LDL) were covalently bound to Sepharose matrices and packed into separate columns. In incubations lacking LTP, greater than 98% of the recovered DAG remained associated with HDLp-L. An unrelated hemolymph storage protein, arylphorin, was unable to catalyze the transfer of DAG between solid-phase lipoproteins. Facilitated transfer of DAG from HDLp-L to LDL was observed when LTP was circulated between the columns. Under these conditions, facilitated transfer occurred at a rate of 2.24 ng of DAG/h (versus 0.16 microgram of DAG/h in the control), and after 16 h greater than 26% of recovered labeled DAG was transferred to LDL. This corresponds to a 14-fold rate enhancement induced by LTP. The LTP-specific transfer of DAG between physically separated lipoproteins demonstrates the ability of LTP to facilitate net lipid transfer via a carrier-mediated mechanism in the absence of a ternary complex involving donor, acceptor, and catalyst. In experiments aimed at assessing the relative contribution of ternary complex formation to DAG transfer, acceptor LDL was circulated with HDLp-L remaining immobilized. Under these conditions, LTP induced a 13-fold rate enhancement from 1.3 to 16.3 micrograms of DAG/h. The similar rate enhancements observed with both lipoproteins bound and only donor bound suggest the overall contribution of ternary complex formation to facilitated lipid transfer is insignificant. The described system should prove useful in mechanistic studies of other transfer proteins as well as studies of transfer of other lipids.     

Transport of lipids in insects

Many insect species are almost completely dependent on lipids for their metabolic needs, although this is usually a function of developmental stage. The primary storage organ is the fat body, which can constitute 50% of the fresh weight of the insect and also acts as the major metabolic center (analogous to the vertebrate adipose tissue and liver). Bathing the fat body (and all other tissues and organs) is the hemolymph, the main functions of which are to transport nutrient substrates to utilization sites and to deliver metabolic wastes to the excretory system. Although neutral lipids are stored as triglycerides, in times of need they appear to be endergonically released into the hemolymph as diglycerides in the majority of insects thus far studied (particularly silkmoths and locusts). Indeed, diglycerides constitute the largest neutral lipid fraction in the hemolymph of silkmoths, locusts, cockroaches, bugs, etc. In the hemolymph the diglyceride is found as a constituent of specific lipoproteins, and one specific lipoprotein class (lipoprotein I; high density lipoprotein) appears to be necessary for the transport of diglyceride from the fat body cell into the hemolymph. This particular lipoprotein is also involved in the transport of cholesterol from the gut into the hemolymph. Thus, lipoprotein I appears to be the major neutral lipid and sterol transport agent in the insects studied and, in addition, plays a regulatory role in the release of both diglycerides and sterols. Hemolymph lipoprotein II (very high density lipoprotein) may be important in providing protein and lipid to the insect ovary during oogenesis. Ecdysone, the polyhydroxy steroidal insect molting hormone, is probably carried "free" in the hemolymph, although reports exist of specific hemolymph-binding proteins in some species. The other major insect growth hormone, juvenile hormone, is transported by hemolymph lipoproteins in silkmoths and locusts and by a lower molecular weight hemolymph protein in the tobacco hornworm.     

Facilitated diacylglycerol exchange between insect hemolymph lipophorins. Properties of Manduca sexta lipid transfer particle

Manduca sexta hemolymph lipid transfer particle (LTP) is a very high density lipoprotein (d = 1.23 g/ml) containing 14% lipid and 5% carbohydrate. Each of three apoprotein components, apoLTP-I (Mr approximately 320,000), apoLTP-II (Mr = 85,000), and apoLTP-III (Mr = 55,000), is glycosylated. Carbohydrate analysis revealed the presence of mannose and N-acetylglucosamine in a ratio of 4.5:1. A native Mr greater than 670,000 was determined by pore limiting gradient gel electrophoresis. Lipid analysis of LTP revealed the presence of phospholipid, diacylglycerol (DAG), free fatty acid, and triacylglycerol. Rabbit polyclonal antibodies directed against LTP were obtained. Anti-LTP serum was employed in experiments which indicated the presence of LTP in larval and adult animals and confirmed that LTP was unrelated to other M. sexta hemolymph proteins and lipoproteins. A quantitative lipid transfer assay measuring facilitated DAG exchange between isolated M. sexta lipoproteins was established. The level of LTP-catalyzed exchange of DAG increased linearly with increasing time and protein during the initial phase of the reaction. Inclusion of anti-LTP serum in the assay inhibited facilitated DAG exchange. Experiments designed to determine if the LTP holoprotein is required for transfer or if a component of LTP is the active principle were performed. Incubation of [3H]DAG labeled high density lipophorin with substrate amounts of LTP resulted in incorporation of labeled DAG into LTP. Subsequent incubation of [3H]DAG-labeled LTP with unlabeled lipophorin resulted in exchange of DAG and the appearance of labeled DAG in lipophorin. Nitrocellulose-bound LTP apoproteins did not facilitate DAG exchange, and pretreatment of LTP with detergents resulted in loss of transfer activity. Extraction of LTP lipids with ethanol/ether also resulted in loss of activity. The results suggest that the lipid component of LTP may be important in the transfer reaction.     

Lipid transfer protein from Manduca sexta hemolymph

A hemolymph lipid transfer protein (LTP) was isolated from the tobacco hornworm, Manduca sexta. LTP catalyzes net lipid transfer between isolated hemolymph lipoproteins in vitro. An isolation procedure employing density gradient ultracentrifugation and gel permeation chromatography produced a purified protein. LTP is a very high density lipoprotein with a particle Mr greater than 500,000. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed that LTP is comprised of two apoproteins: apoLTP-I (Mr approximately 320,000) and apoLTP-II (Mr approximately 85,000). LTP may have a physiological role in altering the lipid content and composition of the major hemolymph lipoprotein, lipophorin.     

Insect lipid transfer particle catalyzes bidirectional vectorial transfer of diacylglycerol from lipophorin to human low density lipoprotein

Insect plasma lipid transfer particle (LTP) catalyzes vectorial net transfer of diacylglycerol (DAG) from Manduca sexta larval high density lipophorin (HDLp-L) to human low density lipoprotein (LDL) producing an LDL of lower density and lipophorin subspecies of higher density. At equilibrium, a stable DAG-depleted very high density lipophorin species (density = 1.25 g/ml) is formed. Electrophoretic analysis of the substrate and product lipoproteins showed that apoprotein exchange or transfer between human LDL and lipophorin did not occur during the lipid transfer reaction. Facilitated net transfer of cholesteryl ester, free cholesterol, and phospholipid occurred to a much lower extent than DAG net transfer, indicating that under these conditions, LDL serves as a sink for lipophorin-associated DAG. This reaction, therefore, provides a method whereby the mass of lipid associated with human LDL can be modified in vitro without alteration of its apoprotein component. The DAG content of LDL increased in a linear manner with respect to LTP concentration and time during the initial phase of the reaction, demonstrating the utility of this system as a quantitative assay method for LTP-mediated net DAG transfer. When [3H]DAG-labeled LDL was prepared and employed in transfer experiments with unlabeled lipophorin, labeled DAG was recovered in the HDLp-L fraction. The amount of labeled DAG recovered in the HDLp-L fraction was dependent on the ratio of LDL to HDLp-L in the reaction. Thus, in this system, LTP-mediated DAG redistribution is bidirectional, suggesting that the final equilibrium distribution of lipid may be dictated by the properties of potential donor/acceptor lipoproteins rather than by an inherent particle substrate specificity of LTP.     

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.     

Free cholesterol is a potent regulator of lipid transfer protein function

This study investigates the effect of altered lipoprotein free cholesterol (FC) content on the transfer of cholesteryl ester (CE) and triglyceride (TG) from very low- (VLDL), low- (LDL), and high-(HDL) density lipoproteins by the plasma-derived lipid transfer protein (LTP). The FC content of VLDL and HDL was selectively altered by incubating these lipoproteins with FC/phospholipid dispersions of varying composition. FC-modified lipoproteins were then equilibrated with [3H] TG, [14C]CE-labeled lipoproteins of another class to facilitate the subsequent modification of the radiolabeled donor lipoproteins. LTP was added and the extent of radiolabeled TG and CE transfer determined after 1 h. With either LDL or VLDL as lipid donor, an increase in the FC content of these lipoproteins caused a concentration-dependent inhibition (up to 50%) of CE transfer from these particles, without any significant effect on TG transfer. In contrast, with HDL as donor, increasing the HDL FC content had little effect on CE transfer from HDL, but markedly stimulated (up to 2.5-fold) the transfer of TG. This differential effect of FC on the unidirectional transfer of radiolabeled lipids from VLDL and HDL led to marked effects on LTP-facilitated net mass transfer of lipids. During long-term incubation of a constant amount of LTP with FC-modified VLDL and HDL, the extent of net mass transfer was linearly related to lipoprotein FC content; a 4-fold increase in FC content resulted in a 3-fold stimulation of the CE mass transferred to VLDL, which was coupled to an equimolar, reciprocal transfer of TG mass to HDL. Since lipid transfer between lipoproteins is integral to the process of reverse cholesterol transport, we conclude that lipoprotein FC levels are a potent, positive regulator of the pathways involved in sterol clearance. FC may modulate lipid transfer by altering the availability of CE and TG to LTP at the lipoprotein surface.     

Deletion of lolB, Encoding an Outer Membrane Lipoprotein, Is Lethal for Escherichia coli and Causes Accumulation of Lipoprotein Localization Intermediates in the Periplasm

Outer membrane lipoproteins of Escherichia coli are released from the inner membrane upon the formation of a complex with a periplasmic chaperone, LolA, followed by localization to the outer membrane. In vitro biochemical analyses revealed that the localization of lipoproteins to the outer membrane generally requires an outer membrane lipoprotein, LolB, and occurs via transient formation of a LolB-lipoprotein complex. On the other hand, a mutant carrying the chromosomal lolB gene under the control of the lac promoter-operator grew normally in the absence of LolB induction if the mutant did not possess the major outer membrane lipoprotein Lpp, suggesting that LolB is only important for the localization of Lpp in vivo. To examine the in vivo function of LolB, we constructed a chromosomal lolB null mutant harboring a temperature-sensitive helper plasmid carrying the lolB gene. At a nonpermissive temperature, depletion of the LolB protein due to loss of the lolB gene caused cessation of growth and a decrease in the number of viable cells irrespective of the presence or absence of Lpp. LolB-depleted cells accumulated the LolA-lipoprotein complex in the periplasm and the mature form of lipoproteins in the inner membrane. Taken together, these results indicate that LolB is the first example of an essential lipoprotein for E. coli and that its depletion inhibits the upstream reactions of lipoprotein trafficking.     

Lipid transfer particle from the silkworm,Bombyx mori,is a novel member of the apoB/large lipid transfer protein family

Lipid transfer particle (LTP) is a high-molecular-weight, very high-density lipoprotein known to catalyze the transfer of lipids between a variety of lipoproteins, including both insects and vertebrates. Studying the biosynthesis and regulation pathways of LTP in detail has not been possible due to a lack of information regarding the apoproteins. Here, we sequenced the cDNA and deduced amino acid sequences for three apoproteins of LTP from the silkworm (Bombyx mori). The three subunit proteins of the LTP are coded by two genes, apoLTP-II/I and apoLTP-III. ApoLTP-I and apoLTP-II are predicted to be generated by posttranslational cleavage of the precursor protein, apoLTP-II/I. Clusters of amphipathic secondary structure within apoLTP-II/I are similar to Homo sapiens apolipoprotein B (apoB) and insect lipophorins. The apoLTP-II/I gene is a novel member of the apoB/large lipid transfer protein gene family. ApoLTP-III has a putative conserved juvenile hormone-binding protein superfamily domain. Expression of apoLTP-II/I and apoLTP-III genes was synchronized and both genes were primarily expressed in the fat body at the stage corresponding to increased lipid transport needs. We are now in a position to study in detail the physiological role of LTP and its biosynthesis and assembly.     

An insect lipid transfer particle promotes lipid loading from fat body to lipoprotein

The role of Manduca sexta lipid transfer particle (LTP) in the transport of lipid from fat body to lipophorin was investigated in vitro. Fat body that contained radiolabeled lipid was incubated with either high density lipophorin or low density lipophorin, and it was shown that lipid was transferred from fat body to lipophorins. The transfer of diacylglycerol was blocked by preincubating fat body with LTP antibody. Furthermore, transfer was restored by the addition of LTP, indicating that LTP promotes the transfer of lipid from fat body to lipophorins. Using lipophorins radio-labeled in their lipid moiety, transfer of lipid from lipophorin to fat body was demonstrated. This transfer was not mediated by LTP. The adipokinetic hormone induced diacylglycerol mobilization from the fat body and the concomitant interconversion of high density lipophorin to low density lipophorin were performed in vitro and were shown to require the presence of LTP.     

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.     

The double life of a bacterial lipoprotein

It has been known for many years that the small lipoprotein Lpp, which is the most abundant protein in E. coli, exists in two forms. The 'bound' form of the protein is tethered to the outer membrane (OM) by its N-terminal lipid moiety and covalently attached to the cell wall by its C-terminal lysine residue. The exact location of the 'free' form, however, has never been determined. In this issue of Molecular Microbiology, Cowles et al. demonstrate that the free form of Lpp is an integral OM protein whose C-terminus is exposed on the cell surface. The new study provides the first example of a lipoprotein that has a dual localization and adds to a growing body of evidence that lipoproteins can span the OM despite the lack of an obvious transmembrane segment. Furthermore, the new results raise intriguing questions about the assembly of both lipoproteins and other types of OM proteins.     

Impact of murine intestinal apolipoprotein A-IV expression on regional lipid absorption, gene expression, and growth

Apolipoprotein A-IV (apoA-IV) is synthesized by intestinal enterocytes during lipid absorption and secreted into lymph on the surface of nascent chylomicrons. A compelling body of evidence supports a central role of apoA-IV in facilitating intestinal lipid absorption and in regulating satiety, yet a longstanding conundrum is that no abnormalities in fat absorption, feeding behavior, or weight gain were observed in chow-fed apoA-IV knockout (A4KO) mice. Herein we reevaluated the impact of apoA-IV expression in C57BL6 and A4KO mice fed a high-fat diet. Fat balance and lymph cannulation studies found no effect of intestinal apoA-IV gene expression on the efficiency of fatty acid absorption, but gut sac transport studies revealed that apoA-IV differentially modulates lipid transport and the number and size of secreted triglyceride-rich lipoproteins in different anatomic regions of the small bowel. ApoA-IV gene deletion increased expression of other genes involved in chylomicron assembly, impaired the ability of A4KO mice to gain weight and increase adipose tissue mass, and increased the distal gut hormone response to a high-fat diet. Together these findings suggest that apoA-IV may play a unique role in integrating feeding behavior, intestinal lipid absorption, and energy storage.     

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.     

Composition and ultrastructure of size subclasses of normal human peripheral lymph lipoproteins: quantification of cholesterol uptake by HDL in tissue fluids

Peripheral lymph lipoproteins have been characterized in animals, but there is little information about their composition, and none about their ultrastructure, in normal humans. Therefore, we collected afferent leg lymph from 16 healthy males and quantified lipids and apolipoproteins in fractions separated by high performance-size exclusion chromatography. Apolipoprotein B (apoB) was found almost exclusively in low density lipoproteins. The distribution of apoA-I, particularly in lipoprotein A-I (LpA-I) without A-II particles, was shifted toward larger particles relative to plasma. The fractions containing these particles were also enriched in apoA-II, apoE, total cholesterol, and phospholipids and had greater unesterified cholesterol-to-cholesteryl ester ratios than their counterparts in plasma. Fractions containing smaller apoA-I particles were enriched in phospholipid. Most apoA-IV was lipid poor or lipid free. Most apoC-III coeluted with large apoA-I-containing particles. Electron microscopy showed that lymph contained discoidal particles not seen in plasma. These findings support other evidence that high density lipoproteins (HDL) undergo extensive remodeling in human tissue fluid. Total cholesterol concentration in lymph HDL was 30% greater (P < 0.05) than could be explained by the transendothelial transfer of HDL from plasma, providing direct confirmation that HDL acquire cholesterol in the extravascular compartment. Net transport rates of new HDL cholesterol in the cannulated vessels corresponded to a mean whole body reverse cholesterol transport rate via lymph of 0.89 mmol (344 mg)/day.     

Overexpression of LolCDE allows deletion of the Escherichia coli gene encoding apolipoprotein N-acyltransferase

Bacterial lipoproteins represent a subset of membrane-associated proteins that are covalently modified with lipids at the N-terminal cysteine. The final step of lipoprotein modification, N-acylation of apolipoproteins, is mediated by apolipoprotein N-acyltransferase (Lnt). Examinations with reconstituted proteoliposomes and a conditional mutant previously indicated that N-acylation of lipoproteins is required for their efficient release from the inner membrane catalyzed by LolA and LolCDE, the lipoprotein-specific chaperone and ABC transporter, respectively. Because Lnt is essential for Escherichia coli, a mutant lacking Lnt activity has not been isolated. However, we report here that lnt-null strains can be constructed when LolCDE is overproduced in strains lacking either the major outer membrane lipoprotein Lpp or transpeptidases that cross-link Lpp with peptidoglycan. Lipoproteins purified from the lnt-null strain exhibited increased mobility on SDS-PAGE compared to those from wild-type cells and could be sequenced by Edman degradation, indicating that lipoproteins in this mutant exist as apolipoproteins that lack N-acylation. Overexpression of Lpp in the lnt-null strain resulted in the accumulation of apoLpp in the inner membrane and caused growth arrest. In contrast to the release of mature Lpp in the presence of LolA and LolCDE, that of apoLpp from the inner membrane was significantly retarded. Furthermore, the amount of lipoproteins copurified with LolCDE was significantly reduced in the lnt-null strain. These results indicate that the affinity of LolCDE for apolipoprotein is very low, and therefore, overexpression of LolCDE is required for its release and sorting to the outer membrane.     

Modulation of lipid synthesis,apolipoprotein biogenesis,and lipoprotein assembly by butyrate

Short-chain fatty acids (SCFAs) are potent modulators of the growth, function, and differentiation of intestinal epithelia. In addition, high-fiber diets may protect against the development of atherosclerosis because of their cholesterol-lowering effects due, in large part, to SCFA production, liver sterol metabolism, and bile acid excretion. Although the small gut plays a major role in dietary fat transport and contributes substantially to plasma cholesterol and lipoprotein homeostasis, the impact of SCFAs on intestinal lipid handling remains unknown. In the present study, the modulation of lipid synthesis, apolipoprotein biogenesis, and lipoprotein secretion by butyrate was investigated in Caco-2 cells plated on permeable polycarbonate filters, which permit separate access to the upper and lower compartments of the monolayers. Highly differentiated and polarized cells (20 days of culture) were incubated for 20 h with 20 mM butyrate in the apical medium. In the presence of [14C]oleic acid, butyrate led to a significant reduction of secreted, labeled triglycerides (27%; P < 0.01) and phospholipids (25%; P < 0.05). Similarly, butyrate significantly decreased the incorporation of [14C]acetate into exported cholesteryl ester (49%; P < 0.005). As expected from these results, with [14C]oleic acid as a precursor, butyrate significantly (P < 0.05) diminished the delivery of radiolabeled chylomicrons and very low-density lipoproteins. In parallel, [35S]methionine pulse labeling of Caco-2 cells revealed the concomitant inhibitory effect of butyrate on the synthesis of apolipoproteins B-48 (28%; P < 0.05) and A-I (32%; P < 0.01). Collectively, our data indicate that butyrate may influence lipid metabolism in Caco-2 cells, thus suggesting a potential regulation of intestinal fat absorption and circulating lipoprotein concentrations.