Cell based screening assays for the triglyceride synthesis pathway

摘要

A method for identifying a compound which modulates the activity of acyl-coA: diacylglycerol acyltransferase comprises the steps of contacting a stable isotope labeled fatty acid with cells in either presence or absence of the compound, extracting the cells with isopropyl alcohol, and determining the level of a stable isotope labeled triglyceride in the presence or absence of the compound; wherein a change in the level of the stable isotope labeled triglyceride indicates that the compound modulates the DGAT activity.

主权项

1. A high-throughput method for identifying a compound which inhibits the in vivo activity of acyl-coA: diacylglycerol acyltransferase (DGAT) in cells, comprising the steps of:
a. contacting a stable isotope labeled fatty acid with said cells in the presence and the absence of said compound under conditions suitable for incorporation into lipids; b. extracting lipids from said cells in a one-step extraction with isopropyl alcohol; c. determining the level of a stable isotope labeled triglyceride in the extract obtained from said cells contacted in the presence of said compound; d. determining the level of a stable isotope labeled triglyceride in the extract obtained from said cells contacted in the absence of said compound; e. comparing the level of the stable isotope labeled triglyceride in the presence of said compound to the level of the stable isotope labeled triglyceride in the absence of said compound; and f. selecting a compound for which the level of the stable isotope labeled triglyceride in the presence of said compound is significantly lower than the level of the stable isotope labeled triglyceride in the absence of said compound thereby identifying the compound which inhibits the in vivo activity of DGAT in said cells; wherein said stable isotope labeled triglyceride is detected by a liquid chromatographic-mass spectrophotometric system; and wherein said cells are differentiated adipocytes or human hepatoma HUH7 cells and the method identifies an inhibitor of DGAT1 activity, or wherein said cells are human breast cancer MCF7 cells and the method identifies an inhibitor of DGAT2 activity.