Clues on the function of Manduca sexta perilipin 2 inferred from developmental and nutrition-dependent changes in its expression.

Cellular triglycerides (TG) are stored in cytosolic lipid droplets (LDs). Perilipins (PLIN) are a group of LD-proteins that play important roles in the assembly and transport of LDs and in TG metabolism. Two members of the PLIN family are found in insects (PLIN1 & 2 or Lsd1 & 2). We have cloned and expressed Manduca sexta PLIN2 (MsPLIN2), and studied developmental and nutritional changes in the expression of PLIN2. Nutritional changes induced fast alterations in PLIN2 mRNA and protein levels in fat body and midgut of the feeding larvae. The relationship observed between PLIN2 expression and TG synthesis in both larval fat body and midgut suggests that PLIN2 is needed when tissues are accumulating TG. However, when the fat body was storing TG at maximal capacity, MsPLIN2 levels declined. This unexpected finding suggests the occurrence of alternative mechanism/s to shield TG from the action of lipases in M. sexta LDs. In addition, it implies that the cellular level of lipid storage could be modulating MsPLIN2 expression and/or degradation. The study also confirmed that MsPLIN2 was most abundant in the adult fat body, which is characterized by a high rate of TG hydrolysis and lipid mobilization. Whether MsPLIN2 is directly involved in lipolysis and/or the secretion of lipids in the fat body of adult of M. sexta is unknown at this time. Nonetheless, the coexistence of high PLIN2 and lipolysis levels suggests a complex role for MsPLIN2. Altogether, we found that MsPLIN2 is needed when the synthesis of glycerides, DG and TG, is active even if the insect is accumulating or consuming TG.

Developmental changes in the protein composition of Manduca sexta lipid droplets.

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.

Mobilization of lipid stores in Manduca sexta: cDNA cloning and developmental expression of fat body triglyceride lipase, TGL.

Fatty acids stored as triglycerides (TG) in the fat body serve as precursor in multiple processes including energy production and synthesis of cellular components. Mobilization of fatty acids from TG depends on the action of lipases. The fat body triglyceride lipase from Manduca sexta, MsTGL, is the only insect lipase that has been purified and characterized, so far. A TGL cDNA from M. sexta fat body encoding a 649 amino acid protein was cloned and its identity confirmed by mass spectrometry and Edman sequencing data of the purified protein. The protein sequence has conserved domains and residues of potential importance for the function and regulation of TGL activity. The expression of TGL and the lipase activity of fat body homogenates were studied in several developmental stages of M. sexta. TG-hydrolase activity of fat body increased as larva grew to the last instar and, then, decreased to minimal levels during pupa stage. Lipase activity was progressively restored in adult insects and reached maximum values at this stage. The fat body lipase activity from adult insects, 1-2 day after emergence, was 9-fold higher than that from 2 to 3 days old 5th-instar larvae. A good correlation was found between the abundance of TGL protein and the lipase activity of fat body homogenates. This correlation and the expression pattern of TGL throughout development are consistent with the notion that TGL is the main fat body TG lipase of M. sexta.

Expression of lipid storage droplet protein-1 may define the role of AKH as a lipid mobilizing hormone in Manduca sexta.

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.