qHTS Assay for Lipid Storage Modulators
NCGC Assay Overview: Storing lipids as a reservoir for energy or the anabolism of elementary metabolites is a common feature of probably all cells and is conserved from bacteria to humans. The universal cellular lipid storage organelle is the so-called lipid storage droplet (LD). Although ubiquitous, LDs share a simple structure composed of a hydrophobic core that harbors the storage lipids, more ..
BioActive Compounds: 19
Depositor Specified Assays
NIH Molecular Libraries Probe Production Centers Network [MLPCN]
NIH Chemical Genomics Center [NCGC]
MLPCN Grant: TBA
Assay Submitter (PI): Beller, Mathias; Max-Planck-Institut fur Biophysikalische Chemie
NCGC Assay Overview: Storing lipids as a reservoir for energy or the anabolism of elementary metabolites is a common feature of probably all cells and is conserved from bacteria to humans. The universal cellular lipid storage organelle is the so-called lipid storage droplet (LD). Although ubiquitous, LDs share a simple structure composed of a hydrophobic core that harbors the storage lipids, which is shielded by a droplet-specific phospholipid monolayer to which proteins are attached. The current model of LD biogenesis involves an incorporation of the lipid core into the membrane leaflets of the endoplasmic reticulum (ER) followed by a subsequent budding-like maturation of a LD, which ultimately pinches off. Once released, LD volume can increase by localized lipogenesis or fusion of existing droplets. Storage lipids are re-mobilized enzymatically by lipase activity. Lipase regulation in the adipocyte is heavily studied and involves multiple components including catecholamine signaling, the LD-associated proteins Perilipin and comparative gene identification-58 (CGI-58) and at least two lipases named hormone sensitive lipase (HSL) and adipocyte triglyceride lipase (ATGL). LD regulation outside of the adipocyte is poorly understood and only few components are known. However, there is an urgent need to learn more about ectopic fat depots as mislocalized storage of lipids, for example in the liver or muscle, is an eminent health problem associated with insulin resistance or the metabolic syndrome. We developed a laser-scanning cytometer assay to enable 1,536-well screening and combined the results of the small molecules screen with an RNAi database based on lipid storage .
NCGC Assay Protocol Summary:
qHTS was performed with embryonic Drosophila S3 cells (Bloomington Drosophila Genomics Resource Center [DGRC]), which showed excellent oleic acid feeding characteristics during RNAi assay development. We dispensed 4 uL of cells at 1.25 x 106 cells/mL into LoBase Aurora COC 1,536-well plates (black walled, clear bottom) with a bottle-valve solenoid-based dispenser (Aurora) to obtain 5,000 cells/well. Triacsin C (Sigma-Aldrich T-4540), an inhibitor of long-chain fatty acyl CoA synthetase that blocks synthesis of triglycerides, was used as a positive control for lipid under-storage. A total of 23 nL of compound solution of different concentrations were transferred to the assay plates using a Kalypsys pin tool equipped with a 1,536-pin array containing 10-nL slotted pins (FP1S10, 0.457-mm diameter, 50.8 mm long; V&P Scientific). One microliter of oleic acid (400 uM) was added, and the plate was lidded with stainless steel rubber gasket lined lids containing pinholes. After 18-24 h incubation at 24 degree celsius and 95% humidity, 4 uL of the dyes BODIPY 493/503 (Molecular Probes) and the Cell Tracker Red CMTPX dye (Molecular Probes) were added to the wells to stain lipid droplets and enumerate cell number, respectively. Fluorescence was detected by excitation of the fluorophores with a 488-nm laser on an Acumen Explorer (TTP Lab Tech). The total intensity in channel 1 (500-530 nm) reflected lipid droplet accumulation. Cells were detected using channel 3 (575-640 nm) with 5-um width and 100-um depth filters. The ratio of the total intensity in PMT channel 1 over total intensity of channel 3 was also calculated. Percent activity was computed relative to an internal control (100% inhibited lipid droplet deposition due to the presence of 20 uM Triacsin C), which was added to 32 wells/plate.
1. Compounds are first classified as being probable inhibitors (compounds decreasing signal), activators (compounds increasing signal), cytotoxic (artifactual results), inactive, or inconclusive based on the ratio and component channel readouts.
2. Active compounds were ranked by efficacy and potency from 99 to 40. Inconclusive outcome compounds were scored based on the phenotype observed: 20 for less conclusive inhibitors, 10 for inconclusive phenotype classification, and 0 for inactive.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)