The lipid droplets (LDs) are intracellular organelles mainly dedicated to the storage and provision of fatty acids. To accomplish these functions the LDs interact with other organelles and cytosolic proteins. In order to explore possible correlations between the physiological states of cells and the protein composition of LDs we have determined and compared the proteomic profiles of lipid droplets isolated from the fat bodies of 5th-instar larvae and adult Manduca sexta insects and from ovaries. These LD-rich tissues represent three clearly distinct metabolic states in regard to lipid metabolism: 1) Larval fat body synthesizes fatty acids (FA) and accumulates large amounts as triglyceride (TG); 2) Fat body from adult insects provides FA to support reproduction and flight; 3) Ovaries do not synthesize FA, but accumulate considerable amounts of TG in LDs. Major qualitative and semi-quantitative variations in the protein compositions of the LDs isolated from these three tissues were observed by MS/MS and partially validated by immuno-blotting. The differences observed included changes in the abundance of lipid droplet specific proteins, cytosolic proteins, mitochondrial proteins and also proteins associated with the machinery of protein synthesis. These results suggest that changes in the interaction of LDs with other organelles and cytosolic proteins are tightly related to the physiological state of cells. Herein, we summarize and compare the protein compositions of three subtypes of LDs and also describe for the first time the proteomic profile of LDs from an insect ovary. The compositions and compositional differences found among the LDs are discussed to provide a platform for future studies on the role of LDs, and their associated proteins, in cellular metabolism.

Adipokinetic hormone (AKH) is the main hormone involved in the acute regulation of hemolymph lipid levels in several insects. In adult Manduca sexta AKH promotes a rapid phosphorylation of “Lipid storage protein-1”, Lsd1, and a concomitant activation of the rate of hydrolysis of triglycerides by the main fat body lipase. In contrast, in the larval stage AKH modulates hemolymph trehalose levels. The present study describes the sequence of a full length Lsd1 cDNA obtained from M. sexta fat body and investigates a possible link between Lsd1 expression and the distinct effects of AKH in larva and adult insects. The deduced protein sequence showed a high degree of conservation compared to other insect Lsd1s, particularly in the central region of the protein (amino acids 211–276) in which the predicted lipid binding helices are found. Lsd1 was absent in feeding larva and its abundance progressively increased as the insect develops from the non-feeding larva to adult. Contrasting with the levels of protein, Lsd1 transcripts were maximal during the feeding larval stages. The subcellular distribution of Lsd1 showed that the protein exclusively localizes in the lipid droplets. Lsd1 was found in the fat body but it was undetectable in lipid droplets isolated from oocytes or embryos. The present study suggests a link between AKH-stimulated lipolysis in the fat body and the expression of Lsd1.

A possible role of cellular uptake and re-secretion of apoA-I in the mechanism of cholesterol efflux induced by apoA-I was investigated using a novel experimental approach. Incubation of adipocytes with a recombinant human apoA-I containing a consensus PKA phosphorylation site, pka-ApoA-I, leads to the appearance of phosphorylated protein in the cell culture medium unambiguously proving cellular uptake and re-secretion of pka-ApoA-I. Phosphorylation of apoA-I is abolished by PKA inhibitors and enhanced by PKA activators demonstrating the specific involvement of PKA. Studies on the concentration dependence of pka-apoA-I phosphorylation and competition experiments with human apoA-I suggest that apolipoprotein uptake is a receptor mediated process. A possible role of apoA-I recycling in the mechanism of cholesterol efflux was investigated by determining the rates of apoA-I induced cholesterol efflux and apoA-I recycling in the presence and in the absence of Brefeldin A (BFA). The studies showed that BFA strongly inhibits cholesterol efflux without affecting the rate of apoA-I recycling. Since BFA affects vesicular trafficking of ABCA1, this study suggests that the interaction of apoA-I with ABCA1 does not mediate apolipoprotein uptake and resecretion. This result suggests that lipidation of apoA-I and apolipoprotein uptake/re-secretion are independent processes. Plasma apolipoprotein A-1 (ApoA-I) plays an important role in the removal of cholesterol from peripheral tissues and, consequently, in the prevention of atherosclerosis dependent cardiovascular disease [1]. The rate of cellular cholesterol removal by apoA-I depends on the plasma membrane levels of cholesterol [2,3] and ATP binding cassette transporter A1, ABCA1 [1,4,5]. The importance of ABCA1 in the process of apoA-I lipidation and the concurrent formation of nascent HDL particles has been demonstrated in numerous studies and is strongly supported by the fact that nonfunctional ABCA1 molecules lead to the development of Tangier's disease [6]. A direct interaction of apoA-I with ABCA1 is in general accepted as a main step required in the transference of cellular lipids to apoA-I [7][8][9][10]. Some studies suggest that ABCA1 promotes the transference of phospholipids alone to apoA-I [7,10,11] whereas other studies suggest that phospholipids and cholesterol are loaded into apoA-I in a concurrent manner [9]. In addition to this uncertainty, it is not clear whether or not the lipidation process requires cellular uptake of apoA-I. Models of ABCA1 dependent lipid loading of apoA-I assuming that lipidation takes place on the surface of the plasma membrane have been recently

Adipose tissue is a major reservoir of cholesterol and, as such, it may play a significant role in cholesterol homeostasis. The aims of this study were to obtain a quantitative characterization of apolipoprotein A-I (apoA-I)-dependent lipid efflux from adipocytes and examine the role of ATPbinding cassette transporter A1 (ABCA1) in this process. The rates of apoA-I-induced cholesterol and phospholipid efflux were determined and normalized by cellular protein or ABCA1 levels. In order to allow a comparative analysis, parallel experiments were also performed in macrophages. These studies showed that apoA-I induces cholesterol efflux from adipocytes at similar rates as from macrophages. Enhancement of the expression of ABCA1 increased the rates of cholesterol efflux from both adipocytes and macrophages. The results also suggested that a non-ABCA1-dependent mechanism could make significant contributions to the rate of apoA-I-dependent cholesterol efflux when the expression levels of ABCA1 are low. Furthermore, the study of the effect of inhibitors of lipid efflux showed that glyburide and brefeldin A, which affect ABCA1 function, exerted strong and similar inhibitory effects on lipid efflux from both adipocytes and macrophages, whereas BLT1, an SRB-I inhibitor, only exerted a moderate inhibition. Overall these studies suggest that ABCA1 plays a major role in apoA-I-dependent lipid efflux from adipocytes and showed high similarities between the abilities of adipocytes and macrophages to release cholesterol in an apoA-I-dependent fashion.

Cellular uptake and resecretion of apoA-I (apoA-I recycling) could be an important factor in determining the circulating plasma levels of apoA-I and/or HDL. Using a novel method to study protein recycling, we have recently demonstrated recycling of apoA-I by adipocytes and suggested that this is a receptor mediated process independent of ABCA1 function. In the present study, it is shown that apoA-I recycling by adipocytes can be blocked by a monoclonal antibody against the β-subunit of ATP synthase, a protein that had been previously identified as an apoA-I receptor. Investigation of the cellular recycling of two other proteins, an apolipoprotein and a small globular protein, showed that recycling of apoA-I is a selective process. The present study also shows that blocking apoA-I recycling has no effect on the rate of apoA-I-induced cholesterol or phospholipid efflux. It is concluded that cellular recycling of apoA-I is a selective process that involves the ectopically expressed β-subunit of ATP synthase. The physiological function of apoA-I recycling remains to be elucidated. However, this study shows that the process of apoA-I uptake and resecretion is not required for apoA-I lipidation.