A self-contained 48-well fatty acid oxidation assay.
The modulation of fatty acid metabolism and especially the stimulation of fatty acid oxidation in liver or skeletal muscle are attractive therapeutic approaches for the treatment of obesity and the associated insulin resistance. However, current beta-oxidation assays are run in very low throughput, which represents an obstacle for drug discovery in this area. Here we describe results for a 48-well beta-oxidation assay using a new instrument design. A connecting chamber links two adjacent wells to form an experimental unit, in which one well contains the beta-oxidation reaction and the other captures CO(2). The experimental units are sealed from each other and from the outside to prevent release of radioactivity from the labeled substrate. CO(2) capture in this instrument is linear with time and over the relevant experimental range of substrate concentration. Cellular viability is maintained in the sealed environment, and cells show the expected responses to modulators of beta-oxidation, such as the AMP kinase activator 5-aminoimidazole carboxamide riboside. Data are presented for different lipid substrates and cell lines. The increased throughput of this procedure compared with previously described methods should facilitate the evaluation of compounds that modulate fatty acid metabolism.
Wang
Xiaojun
X
Metabolic Disease Research, Abbott Laboratories, Abbott Park, IL 60064-3502, USA.
Wang
Rose
R
Nemcek
Thomas A
TA
Cao
Ning
N
Pan
Jeffrey Y
JY
Frevert
Ernst U
EU
D000818:Animals; D001681:Biological Assay / Q000295:instrumentation*; D002210:Caprylates / Q000378:metabolism; D002245:Carbon Dioxide / Q000378:metabolism; D006528:Carcinoma, Hepatocellular / Q000378:metabolism; D045744:Cell Line, Tumor; D002469:Cell Separation; D002470:Cell Survival / Q000187:drug effects; D005227:Fatty Acids / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism; D022781:Hepatocytes / Q000187:drug effects / Q000378:metabolism; D066298:In Vitro Techniques; D008113:Liver Neoplasms / Q000378:metabolism; D010084:Oxidation-Reduction; D010168:Palmitates / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley
D002210:Caprylates; D005227:Fatty Acids; D010168:Palmitates; D002245:Carbon Dioxide; D005947:Glucose; C031492:octanoic acid