Utilisation of respiratory substrates in resting and concanavalin A-activated rat splenocytes

Abstract

The in vitro culture of resting and Concanavalin A activated rat spleen lymphocytes has been carried out with the aim of identifying respiratory substrates used in the generation of additional energy required for lymphocyte activation. Previous work in this field had concentrated on the role of glucose but very little is known of the contribution of alternative substrates. The long term (72h) and short term (24h) culture of rat splenocytes necessitated a prior investigation of the effect of empirical culture conditions onmitogenic stimulation. Cell activation was assessed by the incorporation of [H]-thymidine into DNA in long term cultures, and by [h]-leucine incorporation into protein in short term cultures. Patterns of consumption of glucose, glutamine acetoacetate and D3-hydroxybutyrate were assessed in long term cultures; the uptake of glucose and glutamine was shown to alter on mitogenic stimulation. In addition, the presence of glutamine was shown to be obligatory to long term activation. The ketone bodies showed very little uptake at physiological concentrations; this was not affected by Con A or by prior starvation of the cells. The metabolism of glucose in 72h cultures was shown to be profoundly affected by the prevailing culture conditions. Conventional activation of the cells was demonstrated under conditions which were associated with both aerobic and partially anaerobic glucose metabolism; the consumption of glucose by splenocytes could thus not be used as an index of mitogenic stimulation. The detailed examination of glucose and glutamine metabolism was carried out using radiolabelled substrates in 24h cultures. The complete oxidation of glucose may contribute up to 70% of glucose derived ATP and that of glutamine up to 65% of glutamine derived ATP.