Development of a novel LC/MS method to quantitate cellular stearoyl-CoA desaturase activity.
Stearoyl-CoA desaturase 1 (SCD1) is an enzyme that catalyzes the rate-limiting step in de novo synthesis of monounsaturated fatty acids--mainly oleate and palmitoleate from stearoyl-CoA and palmitoyl-Co A, respectively. These products are the most abundant monounsaturated fatty acids in membrane phospholipids, triglycerides, cholesterol esters. Reports on mice with a targeted disruption of SCD1 gene (SCD1-/-) exhibit improved glucose tolerance and insulin sensitivity compared to wild-type suggesting SCD1 could be a therapeutic target for diabetes and related metabolic diseases. Measurement of SCD1 activity is technically challenging and traditional cell-based SCD1 assay procedure is labor intensive with low throughput. We describe here a novel medium-throughput LC/MS cell-based assay for determining cellular SCD1 activity, facilitating screening of potential SCD1 inhibitor compounds. Confluent HepG2 cells were grown in 24-well plates and incubated with vehicle or an inhibitor followed by incubation with deuterium labeled saturated fatty acid substrates. Total cell lipids were extracted and the conversion of stearate to oleate was measured by liquid chromatography-mass spectrometry. Sterculate, a known inhibitor of SCD1, inhibited the enzyme activity in a dose dependent manner in this assay with a calculated EC(50) of 247 nM. The medium-throughput method described here is an important step towards identifying an inhibitor of SCD1 to treat diabetes and related metabolic diseases.
Dillon
Roslyn
R
Pfizer Global Research, La Jolla Laboratories, 10770 Science Center Drive, San Diego, CA 92121, United States.
Greig
Michael J
MJ
Bhat
B Ganesh
BG
D045744:Cell Line, Tumor; D002477:Cells / Q000201:enzymology*; D002853:Chromatography, Liquid / Q000379:methods*; D003521:Cyclopropanes / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005229:Fatty Acids, Monounsaturated / Q000494:pharmacology; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D019301:Oleic Acid / Q000378:metabolism; D013229:Stearic Acids / Q000378:metabolism; D013230:Stearoyl-CoA Desaturase / Q000037:antagonists & inhibitors / Q000378:metabolism*
D003521:Cyclopropanes; D004791:Enzyme Inhibitors; D005229:Fatty Acids, Monounsaturated; D013229:Stearic Acids; D019301:Oleic Acid; C031183:stearic acid; D013230:Stearoyl-CoA Desaturase; C003345:sterculic acid