Diverse glycerolipid synthesizing enzymes contribute to the daily rhythms in phospholipid synthesis (SAIB 2012)

ACOSTA, VICTORIA; GORNE, LUCAS; SALVADOR, G; PASQUARE SUSANA; GIUSTO, N; GUIDO, M

Mendoza

Congreso; Reunion Anual de SAIB; 2012

SAIB

Circadian clocks distributed all through the body regulate a number of cellular and biochemical processes including those related to lipid metabolism, and the disruption of circadian clocks at the molecular level leads to metabolic disorders including obesity and diabetes. Circadian clocks are even present in immortalized cell lines. In this respect, we have previously shown that [32P]-phospholipid biosynthesis oscillates daily in synchronized fibroblast cultures; however, little is known about the daily regulation of bulk glycerophospholipids (GPLs) synthesis. Here, we found a significant temporal variation in the incorporation of [3H]-glycerol into total GPLs in arrested NIH 3T3 cells synchronized with a 2 h-serum shock, with lowest levels around 28 and 56 h. A significant daily variation was also seen in the activity of GLP- synthesizing and -remodeling enzymes such as phosphatidate phosphohydrolase 1 (PAP-1) and lysophospholipid acyltransferases (LPLAT) respectively with clear antiphase profiles. Strikingly, PAP-1 activity exhibited the highest levels of diacylglycerol (DAG) production at 7, 21 and 42 h post-serum stimulation and the lowest levels at 35 and 56 h, accompanying changes in metabolic labeling. On the contrary, acyltransferases for the different phospholipids displayed maximum activity levels around 21-35 h and minimum values at 14 and 42-49 h post synchronization. We further investigated whether the biosynthesis of phosphatidylcholine (PC), the most abundant GPL, is temporally regulated in synchronized fibroblasts. PC is mainly synthesized through the Kennedy pathway with Choline Kinase (ChoK) and CTP: phosphocholine cytidylyltranferase (CCT) as key regulatory enzymes. PC labeling with [3H]-glycerol or [32P]-phosphate exhibited a significant daily variation with lowest levels at 28-32 and 56 h post stimulation. Maximum levels of radiolabeled PC were accompanied by a narrow increase in CCT activity, which peaked at 6 and 35 h after synchronization, together with a gradual oscillation in ChoK enzyme activity and mRNA expression for the alpha isoform. This enzyme presented the highest levels of activity and expression around 14-21 h post serum shock, and the lowest values around 28-35 h. Overall results demonstrate that synchronized fibroblasts are subject to highly complex levels of metabolic regulation exhibiting temporal variations in the biosynthesis and remodeling of net GPLs and particularly of PC. We found the activation of LPLAT at those times at which the de novo biosynthesis of GPLs and PC is decreased. Our observations suggest the concerted and sequential changes in specific enzyme activities and/or mRNA expression (CCT, ChoK and PAP-1) for the temporal control of PC biosynthesis in immortalized cells upon serum synchronization.