Characterization of the very-low density lipoprotein lipidome exported from primary bovine hepatocytes supplemented choline and methionine.
T. L. Chandler*1, S. J. Erb2, W. A. Myers1, B. A. Barton3, J. W. McFadden1, and H. M. White2, 1Cornell University, Ithaca, NY, 2University of Wisconsin-Madison, Madison, WI, 3Balchem Corp., New Hampton, NY.
Supplementing choline in dairy cattle decreases liver triglyceride (TG), potentially through increased very-low density lipoprotein (VLDL) export. Our objectives were to determine the effect of choline chloride (CC) and D,L-Methionine (DLM) on VLDL TG export from primary bovine hepatocytes and characterize the lipidome of exported VLDL. Hepatocyte monolayers (n = 3 preps) were treated (24 h) with CC (0, 0.01, 0.1, or 1.0 mM) and DLM (0, 0.1, or 0.3 mM; 4×3 factorial) in the presence of 1.0 mM fatty acids. Hepatocytes were then harvested to quantify cellular TG and VLDL isolated from media by size exclusion chromatography. Total VLDL TG was measured by colorimetric assay and untargeted lipidomics using mass spectrometry. Data were normalized to DNA, expressed relative to the 0 mM CC and 0 mM DLM treatment within each prep, and analyzed by PROC MIXED with fixed effect of CC, DLM, the interaction, and random effect of prep. Pearson correlations between cell TG, lipoprotein TG, and lipid species were calculated. Contrasts evaluated for CC were 0 mM vs. (0.01, 0.1, 1.0 mM), linear, and quadratic; and for DLM were 0 mM vs. (0.1, 0.3 mM) and 0.1 vs. 0.3 mM. Cell TG tended to be decreased (P = 0.06) in the presence of CC. Increasing CC quadratically affected (0.96, 1.07, 0.88, 0.98 ± 0.05 arbitrary units (AU); P < 0.01), but DLM did not alter (1.0, 0.98, 0.93 ± 0.05 AU; P = 0.31), VLDL TG. Lipidomics revealed 168 TG, 28 diacylglycerol, 32 phosphatidylcholine (PC), 19 phosphatidylethanolamine, 18 phosphatidylserine, and 10 sphingomyelin species but total of each species was not altered (P > 0.10) by CC or DLM, nor were cell TG correlated (P > 0.10) to these lipids. Although total PC was unaltered, increasing CC linearly decreased (P = 0.05) lyso-PC (LPC; e.g. 18:0), a lipid involved in inflammation. Cell TG were correlated (r = 0.54; P < 0.01) to LPC. Overall, supplying CC and DLM minimally altered the VLDL lipidome. Decreased cell TG and LPC with increased CC should be further examined, especially in the context of liver fat accumulation and inflammation in vivo.