GNMT Expression Increases Hepatic Folate Contents and Folate-Dependent Methionine Synthase-Mediated Homocysteine Remethylation

Table 1 Effects of GNMT expression on cell growth, protein turnover, and folate contents.^a

	Adequate folate^b	Folate restriction^b	% Change	P value
Cell growth^c
Experiment I^b
GNMT−^b	55.3 ± 0.6	57.0 ± 0.5	+3.0 ± 0.8	0.016
GNMT+^b	53.3 ± 1.2	53.6 ± 0.4	0.6 ± 0.7	0.68
P value	0.06	<0.001
% Difference	−3.6 ± 2.3	−5.9 ± 0.6
Experiment II^b
GNMT−	52.5 ± 0.6	73.7 ± 0.3	+40.5 ± 0.5	<0.001
GNMT+	51.1 ± 0.8	69.5 ± 0.7	+36.1 ± 1.3	<0.001
P value	0.07	<0.001
% Difference	−2.7 ± 1.6	−5.4 ± 0.9
Protein turnover^d
GNMT−	0.16 ± 0.01	0.14 ± 0.02	−7.6 ± 13.7	0.40
GNMT+	0.15 ± 0.01	0.15 ± 0.01	0.4 ± 1.6	0.93
P value	0.23	0.72
% Difference	−4.9 ± 5.9	3.3 ± 1.6
Folate (ng/10⁶ cells)
GNMT−	7.6 ± 0.4	2.0 ± 0.5	−73.7 ± 6.9	<0.001
GNMT+	11.4 ± 0.4	4.6 ± 0.5	−54.5 ± 4.7	0.017
P value	<0.001	0.003
% Difference	+49.5 ± 4.9	+ 130.7 ± 23.8

^aAll data are presented as means ± SD (n = 3). Each experiment was repeated at least twice. The P values were calculated by Student t test comparing two cell lines under each condition. The % change was calculated by comparing the mean with those under adequate folate. The % difference was calculated by comparing the mean with negative control cells under the same culture condition. The bolded data were statistically significant (P < 0.05).
^bCell lines and culture conditions. GNMT-: wild-type HepG2 cells transfected with vector only were used as negative control. GNMT+: HepG2 cells transfected with GNMT. Cells were cultured in a modified α-MEM medium under folate abundance (100 nmol/L folinate) or folate restriction (10 nmol/L folinate) for 144 h (Experiment I) or 240 h (Experiment II).
^cCell growth is presented as the doubling time (h) of each cell line under different folate conditions.
^dCells were cultured in α-MEM medium supplemented with l-(5,5,5-²H₃)-leucine (200 µmol/L, 50% of total leucine) for 72 h. Protein turnover was estimated by the enrichments of leucine in the cellular proteins. Protein turnover was measured in cells cultured in low folate for 144 h.

ISSN: 1528-3658