Secondary Assay for lipid storage modulators: AML12 cell LDSA assay for probe SAR
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: 4
NIH Molecular Libraries Probe Production Centers Network [MLPCN]
NIH Chemical Genomics Center [NCGC]
MLPCN Grant: 1 R03 MH085686-01
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 droplet. 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 (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 adapted this assay to AML12 cells to confirm if compounds that showed activity in an S3 cell assay (AID: 2685) were also active in AML12 cells to characterize the translational activity to mammalian cells.
NCGC Assay Protocol Summary:
The assay was performed with AML12 cells, using an oleic acid feeding protocol similar to the S3 assay. We dispensed 4 uL of cells to give 700 cells/well into Greiner bio-one 1,536-well plates (black walled, clear bottom). 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 using a final concentration of 20 uM. The orexin antagonist, SB408124 - previously identified to stimulate lipid over-storage, was used as a positive control for lipid over-storage using a final concentration of 20 uM. 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 was added (400 uM final concentration), 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 2 (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 number of objects in PMT channel 2 over the number of objects in PMT 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.Data is the Fed objects (BODIPY 493/503 fluorophore).
2. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description".
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)