Development of a high-speed capillary electrophoresis assay for the analysis of branched chain amino acids released from 3T3-L1 adipocytes

Abstract

We have developed a high-throughput assay for monitoring branched chain amino acid (BCAA) uptake and release dynamics in 3T3-L1 cells using microdialysis coupled to high speed capillary electrophoresis (CE). Isoleucine, leucine, and valine are important indicators of lipogenesis, as are alanine, glutamate, and glutamine, which are by-products produced through the catabolism of BCAAs. The major focus of this work will be on the development and optimization of a high-speed (high-throughput) CE assay for the separation of fluorescently labeled amino acids; particularly the BCAAs and their downstream metabolites. Development of this assay allows for us to ask important biological questions related to lipogenesis using an in vitro cell model. In combination with bulk culture, release and uptake rates of BCAAs were monitored and assessed as a function of various biological stimuli, including several concentrations of glucose, circulating concentrations of BCAAs, as well as insulin and artificial sweeteners. Changes in release profiles were also monitored as cells progressed through the differentiation cycle in order to see if development stage affects lipogenesis.