Pyruvate carbon flux in response to choline and methionine in bovine primary hepatocytes
T.L. Chandler1, S.J. Erb1, S.J. Bertics1, B.A. Barton2, H.M. White1
1Univ. of Wisconsin-Madison, Madison, WI, 2Balchem Corporation, New Hampton, NY.
Choline and methionine can influence carbon metabolism in bovine hepatocytes. Pyruvate carbon was traced through pathways of gluconeogenesis in order to elucidate the ability of choline or methionine to alter carbon flux. Primary hepatocytes isolated from 4 Holstein calves were maintained as monolayer cultures for 24 h. At 24 h glucose-free media was applied and treatments of choline chloride (CC; 0, 0.01, 0.1, or 1.0 mM) and D,L-Met (DLM; 0, 0.1, or 0.3 mM) were added in a factorial design along with 1.0 mM FA, reflecting the blood FA profile at calving. After 21 h, 1.25 mM [2-14C]pyruvate was added to media and CO2 collected after a 3 h incubation. Cells were harvested to quantify glycogen and 14C enrichment. Parallel treatments were incubated without radiolabeled substrate for 24 h to quantify media glucose. Data were normalized to DNA, expressed relative to a no FA control within each cell prep, and analyzed by PROC MIXED (SAS 9.4) with fixed effects of CC, DLM, their interaction, and random effect of calf. Contrasts evaluated for CC were 0 mM vs. (0.01, 0.1, 1.0 mM) and linear contrast 0.01, 0.1, 1.0 mM. Two contrasts evaluated for DLM were 0 mM vs. (0.1, 0.3 mM) and 0.1 vs. 0.3 mM. Data are reported as least squares means ± SE with differences declared at P≤0.05 and tendencies at P≤0.10. No interactions were detected. Media glucose was decreased by presence of both CC (P<0.01) and DLM (P<0.01), while cellular glycogen was increased by presence of CC (P<0.01), but not DLM (P=0.52). Recovery of pyruvate label as glycogen tended to be decreased (P=0.10) by presence of CC, but not affected (P=0.25) by DLM. Relative enrichment of cellular glycogen by pyruvate label was decreased (P<0.01) by presence of CC and tended (P=0.10) to be decreased by 0.3 mM DLM. Recovery of pyruvate label as CO2 tended to be decreased (P=0.10) by presence of CC, and significantly decreased (P=0.02) by 0.3 mM DLM. Pyruvate carbon spared from oxidation by DLM was not recovered as glycogen carbon. Although CC increased cellular glycogen, decreased 14C enrichment suggests glycogen carbon originated from an alternative glucogenic precursor. Keywords: glucose, glycogen, oxidation
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