TGL-mediated lipolysis in Manduca sexta fat body: possible roles for lipoamide-dehydrogenase (LipDH) and high-density lipophorin (HDLp)

Abstract

Triglyceride-lipase (TGL) is a major fat body lipase in Manduca sexta. The knowledge of how TGL activity is regulated is very limited. A WWE domain, presumably involved in protein-protein interactions, has been previously identified in the N-terminal region of TGL. In this study, we searched for proteins partners that interact with the N-terminal region of TGL. Thirteen proteins were identified by mass spectrometry, and the interaction with four of these proteins was confirmed by immunoblot. The oxidoreductase lipoamide-dehydrogenase (LipDH) and the apolipoprotein components of the lipid transporter, HDLp, were among these proteins. LipDH is the common component of the mitochondrial α-keto acid dehydrogenase complexes whereas HDLp occurs in the hemolymph. However, subcellular fractionation demonstrated that these two proteins are relatively abundant in the soluble fraction of fat body adipocytes. The cofactor lipoate found in typical LipDH substrates was not detected in TGL. However, TGL proved to have critical thiol groups. Additional studies with inhibitors are consistent with the notion that LipDH acting as a diaphorase could preserve the activity of TGL by controlling the redox state of thiol groups. On the other hand, when TG hydrolase activity of TGL was assayed in the presence of HDLp, the production of diacylglycerol (DG) increased. TGL-HDLp interaction could drive the intracellular transport of DG. TGL may be directly involved in the lipoprotein assembly and loading with DG, a process that occurs in the fat body and is essential for insects to mobilize fatty acids. Overall the study suggests that TGL occurs as a multi-protein complex supported by interactions through the WWE domain.

Keywords: Diacylglycerol; Fat body; Lipoamide dehydrogenase; Manduca sexta; Redox; Triglyceride lipase; WWE-domain.

Figure 1. Sequence, Expression and Circular Dichroism of N-terminal region of TGL

A) The amino acid sequence alignment of the N-terminal region of MsTGL with Drosophila WWE-Deltex sequence is shown. The sequence alignment was generated using Alignment Explorer/Muscle (Edgar 2004). Identical residues in the sequences are denoted by asterisks; conservative substitutions are denoted by dots. The locations of α-helices (rectangles) and β-strands (arrows) identified from the crystal structure of WWE-Deltex were adapted from Zweifel et al (Zweifel et al. 2005); B) Coomassie Blue stained SDS-PAGE of the purified 140-residue polypeptide, which was expressed as a fusion protein with thioredoxin, cleaved with enterokinase and then purified; C) Far-UV CD spectrum of the purified N-terminal domain of TGL [amino acids 1–140].

Figure 2. WWE Interacting Proteins Assay

A) Coomassie blue stained gel of the soluble proteins eluted from by Ni-sepharose beads containing Trx-WWE fusion protein (lane 1) or Trx (lane 2). Fat body soluble proteins were incubated with 12.5nmol of Trx-WWE bound to Ni-sepharose beads for 2h and the resin was subsequently washed four times with buffer. Proteins were eluted in one step by incubation with 1M imidazole. Co-eluted proteins were analyzed by SDS-PAGE in 4–20% acrylamide gradient gels (lane 1). Control experiment was carried out using 12.5nmol of Trx bound to Ni-sepharose beads (lane 2). Protein markers (BenchMark protein ladder) are shown in lane M (see Materials). B) Western blotting: aliquots of eluted proteins from test (lane 1) and control (lane 2) resins were separated by SDS-PAGE in 4–20% acrylamide gel and transferred to nitrocellulose. Blots were analyzed by immunodetection for the following proteins: ApoLp-I and II, LipDH, GST and TGL as indicated in Materials and Methods.

Figure 3. Co-immunoprecipitation of TGL with anti-HDLp antibodies

Fat body soluble proteins were incubated with anti-HDLp antibody for 2h and immunoprecipitated using protein-A-agarose beads preblocked with BSA. After washing the beads four times with PBS the associated-proteins were separated by SDS-PAGE gel in 4–15% acrylamide gel (lane 1), and analyzed by western blotting using anti-HDLp (lane 2) and anti-TGL antibodies (lane 3). The arrow indicates IgG. ApoLp I/II and TGL bands are also shown in lane 2 and 3, respectively.

Figure 4. Subcellular Localization of LipDH in M.sexta Fat Body

Subcellular fractions of adult fat body homogenates were separated by SDS-PAGE and analyzed by western blotting using anti-LipDH antibody. Approximately 10 μg (lanes “a” and “d”) and 30 μg (lanes “b” and “c”) of total proteins were loaded in the corresponding lane. Lane a: lipid droplets; lane b: cytosol; lane c: 100,000g pellet (P100); lane d: 20,000g pellet (P20). A representative western blotting result is shown.

Figure 5. Effect of DTT, GSH, GSSG and NEM on TGL Activity

Partially purified TGL was pre-incubated with the indicated concentrations of DTT, GSH, GSSG (A), NEM (B) for 30 min on ice prior to measure the lipase activity. Lipase activity was expressed as a fold change relative to control (no addition). Values are the mean ± SEM of three independents experiments.

Figure 6. Effect of Carmustine (A) and Auranofin (B) on lipase activity

Fat body soluble fractions were pre-incubated with the indicated concentrations of inhibitor for 30 min on ice prior to measure lipase activity. Experiments were conducted in the presence and absence of 10 mM DTT. Lipase activity was expressed as a fold change relative to control (no inhibitor). Values are the mean ± SEM of three independent experiments. Significant differences determined by one way ANOVA are shown with asterisks. [***] indicates p<0.001 and [*] indicates p <0.05.

Figure 7. Effect of AKH on lipase activity in the presence and absence of reducing agents

Freshly dissected fat bodies from two insects that were injected with PBS (Control, bars a–b) or AKH (AKH, bars c–d) were pooled and homogenized. Fat body soluble fractions were tested for lipase activity in the absence (−) and presence (+) of 10 mM DTT. Lipase activity was expressed in nmol TG hydrolyzed/min.mg of total protein. Values are the mean ± SEM of three independents experiments. P values are: p^ab >0.05; p^ac<0.001; p^ad<0.001; p^bc <0.05; p^bd <0.001; p^cd <0.001.

Figure 8. Subcellular Distribution of Lipophorin, DG and TG

Fat body homogenate from adult male insects was subjected to ultracentrifugation in a sucrose gradient. The gradient was fractionated into seven fractions. The distribution of ApoLp-I/II, DG and TG is shown in panel A, B and C, respectively. Top panel shows a representative western blot depicting the distribution of ApoLp-II among fractions. For this purpose, 0.6% of fraction 1 to 6, and 0.1% of fraction 7 were loaded in 4–20 % acrylamide gel, transferred to nitrocellulose and analyzed by western blotting. The concentration of sucrose of the fractions is given in g/100ml. Panel A graph comes from the densitometry of the blot; panel B and C were obtained after separation of lipids by TLC as described under Materials and Methods. After visualizing lipids spots with iodine, plates were scanned and lipids quantified with AlphaEaseFCTM software. TG and DG contents were expressed as percentage of total neutral lipid. Two independent experiments were carried and Fig 8 shows a representative result.

Figure 9. Effect of Lipophorin on the Lipase Activity of TGL

A) The lipase activity of partially purified lipase was determined by incubating 25 nmoles of [³H]-triolein-Triton X-100 with 15μg of TGL in the presence of increasing amounts of lipophorin (HDLp; δ=1.15g/cm³) for 30 min. The formation of individual products (DG, MG) was determined after analyzing the reaction products by TLC coupled to scintillation counting. A) Data representative of one experiment are shown in panel A. Data were expressed in nmoles and are represented as the mean ± SEM of three determinations ; B) The effect of lipophorin concentration on the molar ratio DG to MG produced were calculated from the data shown in panel A.