Daily fluctuations in the biosynthesis of phosphatidylcholine and activity of its key enzyme CTP: phosphocholine cytidylyltranferase in synchronized cultures of fibroblasts

ACOSTA-RODRÍGUEZ VA; SEBASTIAN MARQUEZ; GUIDO ME.

Cholula

Simposio; XI Simposio Latinoamericano de Cronobiología(X LASC); 2011

SOCIEDAD LATINOAMERICANA DE CRONOBIOLOGIA

An open question in biology is how the lipid components of the biological membranes are synthesized and assembled to maintain membrane homeostasis and diverse cellular processes. Circadian clocks regulate diverse biochemical functions, whereas cell cultures make up a model of peripheral oscillators to investigate metabolic oscillations. We previously reported that phospholipid biosynthesis oscillates in synchronized fibroblast cultures; however, the way phosphatidylcholine (PC) synthesis is daily regulated in cell cultures, is unknown. PC is mainly synthesized through Kennedy’s pathway with CTP: phosphocholine cytidylyltranferase (CCT) as key enzyme. In mammals, there are two genes encoding for CCT: Pcyt1a that encodes the CCTa protein from alternative transcripts CCTa1 and CCTa2 and Pcyt1b gene encodes the CCTb2 and CCTb3 proteins from differentially alternative spliced mRNA CCTb2 and CCTb3. Here, we investigated possible temporal changes in PC biosynthesis and CCT activity and expression in quiescent NIH 3T3 fibroblasts after a 2 h-serum synchronization. We found that PC labelled with [3H]glycerol or [32P]phosphate in the cultures exhibits a daily variation with levels peaking at 4-6 h after serum stimulation and decreasing by 29-32 h. Significant daily variations were also seen in CCT activity with higher levels at 6 and 35 h. Moreover, CCT expression across time for the most abundant isoforms (CCTa1 and CCTb2) at mRNA and protein levels displayed complex pattern expression. Both CCTa1 transcript and protein peaked at 3 h post-stimulation, while CCTb2 mRNA was higher at 9-12 h. We also detected increased levels of CCTb isoforms during 9-18 h using an antibody that recognized both CCTb2 and CCTb3 proteins. Results demonstrated that synchronized fibroblasts exhibit temporal variations in PC biosynthesis which could be due, at least in part, to changes in CCT activity and/or to a concerted and differential expression of different CCT isoforms