Measurement of fatty acid and glucose oxidation by fetal heart cells in monolayer tissue culture

Abstract

We have utilized a method to investigate substracte oxidations of embryonic chick heart cells growing in monolayer culture. The cells are growth in small capped Falcon flasks in F-12 media supplemented with fetal calf serum and chick embryo extract. For the assay the cells are washed with buffer to remove the media and are then incubated with 14C-palmitate for 1 hour at 37°. After perchloric acid is injected through a serum cap to terminate the reaction, Hyamine-OH is injected into a polypropylene well fixed to the serum cap to trap the evolved 14CO2 which can then be counted. The oxidation of palmitic acid is very active and further stimulated if carnitine is included in the media. Palmitic acid oxidation is decreased by over 75% if the cells are scraped from the flasks prior to the assay perhaps due to alternations of the surface properties of the cells. The specific activity of palmitic acid oxidation is constant regardless of days in culture or initial plating density. In contrast, the specific activity of glucose oxidation is high during cell proliferation and markedly decreases as the cells plateau. Scraping the cells from the flask had little effect on oxidation. The decrease in glucose oxidation is associated with a stiking fall in the raio of glucose-1-14C to glucose-6-14C oxidation indicating a shift away from the hexose monophosphate shunt. Insulin stimulates glucose oxidation by 13 day embryonic heart cells when the cells are proliferating and show high specific activity for glucose oxidation. This method provides a rapid and convenient system for investigation of substrate oxidations in tissue culture.