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.

Lipase Activity in the Larval Midgut of Rhynchophorus palmarum: Biochemical Characterization and the Effects of Reducing Agents

“…The triglyceride lipases from the fat bodies of M. sexta showed the same effects, and the authors explained that GSSG would form a mixed disulfide with the lipase thiol groups. This explained the decreased of its activity, similar to that found with other proteins [ 18 ].…”

“…The thiol groups are important for the lipase activities in the fat bodies of Manduca sexta [ 18 ]. In order to investigate the importance of thiol groups in R. palmarum , homogenates were incubated in different concentrations of GSH, GSSG, and DTT.…”

“…Digestion of ingested food is a process by which nutrients are acquired by insects, and if we understand the physiology of insect digestion, it will be possible to establish possible targets for insect control [ 18 ]. R. palmarum is one of the most important pests of commercial palm plantations [ 10 ].…”

“…The lid, which covers the active site, rotates to allow the interaction with the substrate. These Cys residues are sensitive to thiols and are involved in the mechanism of lipase activation [ 18 ]. The presence of thiol groups, via increasing concentrations of GSH, GSSG, and DTT, was observed to affect lipase activities, indicating that lipase activities are sensitive to redox status ( Figure 5 B).…”

Lipid Droplets as Signaling Platforms Linking Metabolic and Cellular Functions

“…Additional information on the protein compositions of the LDs was previously reported. 10 For comparison, the protein profile of Manduca sexta low density lipoprotein purified by ultracentrifugation in a density gradient 180 is shown in the lane labeled LDLp. ApoLp-I, -II and -III refer to the protein components of the insect low density lipoprotein.…”