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

Abstract

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.

Figure 1. Nucleotide and deduced amino acid sequence of Lsd1 from M. sexta

cDNA nucleotide (1–1704) is shown above the deduced amino acid sequences (1–373). Amino acid residues are aligned with the first nucleotide of each codon. The stop codon TGA is marked by an asterisk. The amino acid sequence underlined represents the matched peptides obtained from the Maldi-tof analysis of the tryptic digest of the gel excised protein. The possible polyadenylation signals are shown underlined. MsLsd1 cDNA sequence has been deposited to GenBank (Accesion number EU809925).

Figure 2. Alignment of Lsd1 deduced amino acid sequences of M. sexta, B. mori and D. melanogaster.

MS (M. sexta, accession number EU809925), BM (B. mori, accession number NP_001040143) and DM (D. melanogaster, accession number NP_732904). Identical (*) and similar (.) residues are indicated underneath the sequences. The alignment was performed using the program CLUSTALW available at www.ch.emb.net.org. The highly conserved region that is predicted to be involved in lipid binding (Arrese et al., 2008) is framed.

Figure 3. Subcellular localization of Lsd1 in adult M. sexta in basal and stimulated (AKH) conditions

[³²P]-Fat body homogenates were collected at 0 and 10 min after AKH treatment. Lipid droplets (lanes 1-2), cytosol (lanes 3–4), and membranes (lanes 5–6) were isolated from control and AKH treated fat bodies, and subjected to SDS-PAGE in 10% acrylamide gel. A) Coomassie Blue- stained gel. B) Autoradiogram of the gel shown in A. C) Western blot analysis using anti-Lsd1 polyclonal antibody preparation. Control samples are represented in lanes 2, 4, and 6, whereas AKH samples are represented in lanes 1, 3, and 5, respectively. Approximately 30–35 μg of total protein was loaded into each lane.

Figure 4. SDS-PAGE and immunoblot analysis of Lsd1 protein levels in the lipid droplets purified from fat body of M. sexta during development

SDS-PAGE gel stained with Coomassie Blue (A) and immunoblot (B) of M. sexta lipid droplets collected from different developmental stages. Lane M) Molecular weight marker; 1) 5^th instar (2^nd day); 2) 5^th instar (5^th day); 3) Wanderer (2^nd day); 4) Pupa (17^th day) and 5) Adult (2^nd day). Approximately 30 μg total protein was loaded in two identical gels. The second gel was used for Western blot (B).

Figure 5. SDS-PAGE and immunoblot analysis of Lsd1 level in the lipid droplets purified from adult female

M. sexta. SDS-PAGE gel stained with Coomassie Blue (A) and immunoblot (B) of M. sexta lipid droplets collected from fat body (lane 1), oocytes (lane 2) and embryos (lane 3). Approximately 50 μg total protein was loaded in two identical gels. The second gel was used for Western blot.

Figure 6. MsLsd1 transcript levels during development

The relative level of the Lsd1 transcript was determined by RT–PCR in a duplex PCR that was performed using primers specific for MsrpS3 (M. sexta ribosomal protein S3) and MsLsd1. Amplification step of the duplex PCR included 22 cycles of 30 s denaturing at 94 °C, 30 s annealing at 56°C, and 1 min extension at 72 °C. The primer concentrations and cycling conditions were optimized in preliminary experiments to avoid saturation. RT-PCR products were separated on 2% agarose gels containing 0.5 μg/ml ethidium bromide and photographed over UV light. A representative gel image is shown. The expected sizes for Lsd1 and rpS3 PCR products are 497 and 415 bp, respectively. Two independent sets of total RNA were independently analyzed at least by duplicate.