Mobilization of Lipid Stores in Manduca Sexta: Role of Adipose Triglyceride Lipase

Abstract

Triglycerides (TG) stored in lipid droplets (LDs) are the main energy reserve in all animals. The mechanism by which animals mobilize TG is complex and not fully understood. Several proteins surrounding the LDs have been implicated in TG homeostasis such as mammalian Perilipin A and insect lipid storage proteins (Lsd). Lipid storage protein 1 (Lsd1) is a conserved insect protein and plays significant roles in the regulation of TG metabolism. Most of the knowledge on LD-associated proteins comes from studies using cells or LDs leaving biochemical properties of these proteins uncharacterized. Here we describe the purification of recombinant MsLsd1, its reconstitution in lipoprotein particles and the mutagenesis studies in putative phosphoryation sites and conserved region of Lsd1. Moreover, mobilization of 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. This study also describes another enzyme, adipose triglyceride lipase (ATGL), and investigates a possible link between ATGL expression and nutrition levels in M.sexta. The ATGL cDNAs from M. sexta fat body encoding a 64KDa protein were cloned. Northern blot analysis detected two bands corresponding to the 3.9 and 2.4 kb transcripts, respectively. The protein sequence has the consensus lipase catalytic motif (GxSxG) and conserved "patatin-like"domain which is a key signature of ATGL enzymes isolated from other organisms. ATGL is mostly associated to the lipid droplet. Sf9 cells over-expressing the MsATGL showed lower content of cellular TG and a higher TG hydrolase activity of purified protein indicating that it is a lipase. MsATGL is up-regulated during the physiological non-feeding periods but lower than TGL with the exception of 3rd-day pre-pupal. Both levels of transcripts and expression of ATGL were dramatically up-regulated by starvation in a time-dependent manner indicating that ATGL is highly sensitive to the nutritional status of animals and may play a key role under starvation conditions. Thus, ATGL and TGL coordinately catabolize stored TGs in M.sexta. These studies provide the starting point for future studies on the mechanism and function of MsLsd1 and ATGL.