Abstract
The hydrolysis of triglyceride (TG) stored in the lipid droplets of the insect fat body is under hormonal regulation by the adipokinetic hormone (AKH), which triggers a rapid activation cAMP-dependent kinase cascade (protein kinase A (PKA)). The role of phosphorylation on two components of the lipolytic process, the TG-lipase and the lipid droplet, was investigated in fat body adipocytes. The activity of purified TG-lipase determined using in vivo TG-radiolabeled lipid droplets was unaffected by the phosphorylation of the lipase. However, the activity of purified lipase was 2.4-fold higher against lipid droplets isolated from hormone-stimulated fat bodies than against lipid droplets isolated from unstimulated tissue. In vivo stimulation of lipolysis promotes a rapid phosphorylation of a lipid droplet protein with an apparent mass of 42-44 kDa. This protein was identified as "Lipid Storage Droplet Protein 1" (Lsdp1). In vivo phosphorylation of this protein reached a peak approximately 10 min after the injection of AKH. Supporting a role of Lsdp1 in lipolysis, maximum TG-lipase activity was also observed with lipid droplets isolated 10 min after hormonal stimulation. The activation of lipolysis was reconstituted in vitro using purified insect PKA and TG-lipase and lipid droplets. In vitro phosphorylation of lipid droplets catalyzed by PKA enhanced the phosphorylation of Lsdp1 and the lipolytic rate of the lipase, demonstrating a prominent role PKA and protein phosphorylation on the activation of the lipid droplets. AKH-induced changes in the properties of the substrate do not promote a tight association of the lipase with the lipid droplets. It is concluded that the lipolysis in fat body adipocytes is controlled by the activation of the lipid droplet. This activation is achieved by PKA-mediated phosphorylation of the lipid droplet. Lsdp1 is the main target of PKA, suggesting that this protein is a major player in the activation of lipolysis in insects.
Highlights
(TG),1 which constitute Ͼ90% fat body lipids
The hydrolysis of triglyceride (TG) stored in the lipid droplets of the insect fat body is under hormonal regulation by the adipokinetic hormone (AKH), which triggers a rapid activation cAMP-dependent kinase cascade (protein kinase A (PKA))
Lipid Storage Droplet Protein 1” (Lsdp1) is the main target of PKA, suggesting that this protein is a major player in the activation of lipolysis in insects
Summary
Materials—[32PO4]Orthophosphate and [␥-32PO4]ATP were purchased from MP Biochemicals (Irvine, CA). Lipid droplets of two insect fat bodies were resuspended in 0.5 ml of buffer. The lipid droplet-associated proteins were separated by SDS-PAGE on 10% gels according to Laemmli but in sample buffer containing 6% (w/v) SDS. For peptide mass fingerprinting of 42 and 44 kDa, lipid droplet-associated proteins were separated on 10% SDS-PAGE and the bands visualized by Coomassie Blue staining, excised, minced, and destained using 100% acetonitrile followed by four washes with water. Aliquots of lipid droplets resuspended in TG-lipase reaction buffer were freshly stained with Oil Red O and were observed under the microscope. After 20 min of incubation at room temperature, the reaction was terminated by the addition of electrophoresis sample buffer and analyzed by SDS-PAGE on 4 –20% acrylamide gels followed by autoradiography. Statistics—Statistical comparisons were made by the Student’s t test. p Ͻ 0.05 was considered to be significant
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