Polyunsaturated fatty acids stimulate phosphatidylcholine synthesis in PC12 cells

Abstract

The synthesis of phospholipids in mammalian cells is regulated by the availability of three critical precursor pools: those of choline, cytidine triphosphate and diacylglycerol. Diacylglycerols containing polyunsaturated fatty acids (PUFAs) apparently are preferentially utilized for phosphatide synthesis. PUFAs are known to play an important role in the development and function of mammalian brains. We therefore studied the effects of unsaturated, monounsaturated and polyunsaturated fatty acids on the overall rates of phospholipid biosynthesis in PC12 rat pheochromocytoma cells. Docosahexaenoic acid (DHA, 22:6n-3), eicosapentaenoic acid (EPA, 20:5n-3) and arachidonic acid (AA, 20:4n-6) all significantly stimulated the incorporation of (14)C-choline into total cellular phospholipids. In contrast, monounsaturated oleic acid (OA) and the saturated palmitic (PA) and stearic (SA) acids did not have this effect. The action of DHA was concentration-dependent between 5 and 50 microM; it became statistically significant by 3 h after DHA treatment and then increased over the ensuing 3 h. DHA was preferentially incorporated into phosphatidylethanolamine (PE) and phosphatidylserine (PS), while AA predominated in phosphatidylcholine (PC).

Fig. 1. Effect of fatty acids on phospholipid synthesis in PC12 cells

Cells were incubated for 4 hrs with fatty acids (20 μM) and, during the last 2 hrs of incubation, with ¹⁴C-choline (3 μCi/ml). Radioactivity incorporated into total cell phospholipids was measured. Data are the means ± SEM of 6 dishes and represent (A) one of 3 or (B) one of 2 independent experiments. *, P<0.05; **, P<0.01.

Fig. 1. Effect of fatty acids on phospholipid synthesis in PC12 cells

Cells were incubated for 4 hrs with fatty acids (20 μM) and, during the last 2 hrs of incubation, with ¹⁴C-choline (3 μCi/ml). Radioactivity incorporated into total cell phospholipids was measured. Data are the means ± SEM of 6 dishes and represent (A) one of 3 or (B) one of 2 independent experiments. *, P<0.05; **, P<0.01.

Fig. 2. DHA effect on phosphatidylcholine level in PC12 cells

Replicate dishes were incubated for 4 hrs in the absence or presence of 20 μM DHA. Phospholipids were extracted, separated and quantitated as described in Experimental Procedures. Results are shown as the means ± SEM of 6 dishes from one of two experiments. ***, P<0.001.

Fig. 3. DHA dose and phosphatidylcholine synthesis in PC12 cells

Cells were incubated for 4 hrs with the indicated doses of DHA (2.5, 5, 10, 50 μM). Incorporation of ¹⁴C-choline into total phospholipids during the last 2 hrs of incubation was measured. Data show the means ± SEM of 6 dishes, representative of 2 experiments. *, P<0.05; **, P<0.01.

Fig. 4. Time course of DHA-stimulated phospholipid synthesis

Replicate dishes of PC12 cells were incubated with or without 20 μM DHA for the time periods indicated and incorporation of ¹⁴C-choline into phospholipids during a 2 hr period was measured. For incubations less than 2 hrs, the addition of ¹⁴C-choline preceded addition of DHA. Results are from 6 dishes/group and are expressed as the ratio of DHA-treated/untreated cells. The measurement in each group is [¹⁴C-phospholipid], (dpm/μg DNA). The experiment was repeated twice. ***, P<0.001.

Fig. 5. Incorporation of AA and DHA into phospholipid esters

Replicate dishes of PC12 cells were incubated for 3 hrs with 0.1 μCi ³H-AA or ¹⁴C-DHA and with 10 μM of the corresponding nonradioactive fatty acids. Phospholipids were extracted, separated by TLC and the radioactivity in individual spots was measured. Quantitites of PC, PS, PI and PE were determined by phosphate analysis. Data are the means ± SEM of 5 dishes and are expressed as dpm ³H or ¹⁴C incorporated/nmol PO₄. ***, P<0.001 for differences between AA and DHA-treated groups.