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Small molecule inhibitors of fatty acid uptake in 3T3‐L1, primary human adipocytes, INS‐1E and C2C12 cell lines
Author(s) -
Jesch Elliot Denver,
Wang Zhigang,
Black Paul N,
DiRusso Concetta C
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.691.1
Subject(s) - cell culture , fatty acid , biochemistry , c2c12 , chemistry , cytotoxicity , 3t3 cells , microbiology and biotechnology , biology , transfection , in vitro , myogenesis , genetics , gene
Previous work in this lab identified 5 chemically distinct small molecules that inhibit the uptake of fatty acid (C1‐BODIPY‐C12) through a series of screenings in yeast expressinghomo sapiens fatty acid transport protein (FATP) 2 and Caco2 cells. Subsequently, we looked to examine the effectiveness and toxicity of each compound in a variety of cells that accumulate lipid. We examined two adipocyte cell lines (mouse 3T3‐L1 and primary human), one pancreatic beta cell line (INS‐1E) and one skeletal muscle cell line (mouse C2C12). Cells were seeded into 96 well (black wall/optically clear bottom) plates and differentiated or allowed to incubate until firmly attached. Cells were subsequently starved for one hour in serum free MEM, one hour incubation with a dilution series of each compound (1nM‐1mM, range dependent on individual cell line), a 5–15 minute incubation with the fluorescent fatty acid C1‐BODIPY‐C12 and finally measured fluorescence intensity using 485/20 nm excitation and 530/20 nm emission filters. Sigmoid dose‐response curves for C1‐BODIPY‐C12uptake were generated in each cell line and EC50 values were from the low nM to high μM range, depending on the particular compound and cell line. The compounds were determined to benon‐toxic to each cell line by utilizing the MTT cytotoxicity assay. Specificity to fatty acid uptake was confirmed by measuring acyl‐CoA synthestase activity and glucose uptake. [Supported by NIH DK071076 ]