This was accomplished by measuring the incorporation of [3H]acetate into TG (Fig. 2A), and in vivo hepatic TG secretion following inhibition of VLDL metabolism with poloxamer (P-407) (Fig. 2B). We also determined ketone bodies in serum (Fig. 2C) and the in vitro secretion of acid-soluble metabolites (Krebs cycle metabolites and ketones) (Fig. 2D), as a measure of FA β-oxidation. Whereas lipogenesis and FA β-oxidation were barely altered in hepatocytes from Gnmt−/− mice (Fig. 2A,C,D), the hepatic TG secretion rate in GNMT-depleted livers was elevated compared to livers from WT animals (Fig. 2B). Consistent with these studies, a comprehensive gene expression analysis showed that, overall, the expression

of genes that supply NADPH and acyl-CoA find more for lipid synthesis was unaltered in mice FK506 solubility dmso without GNMT (Fig. 2E). Despite the marked hepatic steatosis, mice without GNMT did not show insulin resistance or changes in serum FA concentrations (Supporting Fig. 1a,b). Depletion of GNMT in mice did not alter food intake or body weight (Supporting Fig. 1c,d). The greater liver weight in Gnmt−/− mice was not accompanied by differences in body weight, which may be explained by the reduced mass of the white adipose tissue in these animals (Supporting Fig. 1d-f). Based on the results depicted in Fig. 2, which demonstrate that Gnmt−/− mice have increased lipid secretion without affecting lipid synthesis or

oxidation, it is not obvious how to explain the presence of fatty livers in these animals. We reasoned that an elevation of SAMe in Gnmt−/− mice

would activate the flux from PE to PC via PEMT, which would lead to increased PC catabolism and the corresponding augmentation of hepatic DG and TG production (Fig. 2). To confirm this hypothesis, we measured the incorporation of [3H]ethanolamine into PE and PC MCE in hepatocytes isolated from 3-month-old Gnmt−/− mice and calculated the radioactivity incorporated into PC as a percentage of the radiolabel incorporated into PC+PE (Fig. 3A). Because PC formed via PEMT primarily contains long-chain polyunsaturated FA (PUFA), such as docosahexaenoic acid (22:6n-3), whereas PC synthesized by the CDP-choline route do not, we also determined the PC(22:6n-3) to total PC ratio in GNMT-depleted and WT livers as a marker of hepatic PEMT activity.[21] Given that PEMT activity is primarily located in the endoplasmic reticulum,[22] we measured the content of PE and PC in whole liver microsomes (Fig. 3B,C). As shown in Fig. 3, high SAMe levels in Gnmt−/− hepatocytes associated with a 2.5-fold increase in the flux from PE to PC (P < 0.001) (Fig. 3A), and an increase in the PC(22:6)/PC ratio (from 0.18 ± 0.005 in WT to 0.25 ± 0.005 in GNMT-depleted livers, P = 3.23E-06). Also as predicted, the content of PE was reduced ∼2-fold in microsomes isolated from GNMT-depleted livers (P < 0.05), whereas the amount of PC was increased 2-fold (P < 0.05) (Fig. 3B,C).