Hepatic gluconeogenesis is differentially altered by choline and methionine in bovine primary hepatocytes
T.L. Chandler1, S.J. Bertics1, B.A. Barton2, H.M. White1
1Univ. of Wisconsin-Madison, Madison, WI, 2Balchem Corporation, New Hampton, NY.
Coordinated expression patterns of genes controlling gluconeogenesis in primary hepatocytes suggested increased capacity for gluconeogenesis with increasing choline, but not methionine. The objective of this experiment was to quantify glucose export and cellular glycogen in primary bovine hepatocytes exposed to increasing concentrations of choline chloride (CC), D,L-Met (DLM), and added fatty acids (FA). Primary hepatocytes isolated from 3 Holstein calves were maintained as monolayer cultures for 24 h prior to treatment with CC (61, 128, 2028, 4528 μM) and DLM (16, 30, 100, 300 μM), with or without a 1 mM FA cocktail in a 4×4×2 factorial design. Treatments were applied in triplicate to basal media with 5.5 mM glucose and 1.25 mM sodium pyruvate. After 24 h, media was collected to quantify glucose, and cells were harvested to isolate glycogen and quantify DNA. Total glucose, comprised of media glucose and glucose exported from cells, and cellular glycogen were normalized to corresponding total DNA to account for differences in cell plating density between calves, prior to averaging the normalized values within triplicates. Data were expressed relative to the lowest CC and DLM treatment without FA within each cell prep before data were analyzed using PROC MIXED of SAS 9.4 with linear and quadratic contrasts in a model with fixed effect of treatment, corresponding interactions, and random effect of calf. Interactions were not significant; therefore, only main effects are discussed. Fatty acid treatment did not affect media glucose (P=0.98) or cellular glycogen (P=0.29). Increasing CC linearly decreased (P=0.0019) relative glucose in media by up to 13.5%, and linearly (P>0.0001) and quadratically (P<0.0001) increased relative cellular glycogen by up to 26.7%. Media glucose (P=0.86) and cellular glycogen (P=0.69) were not affected by DLM. Increased cellular glycogen with increasing CC, and a lack of change in media glucose or cellular glycogen with increasing DLM, supports previous gene expression data. Differential effects of CC on cellular glucose export and cellular glycogen should continue to be investigated. Keywords: glucose, glycogen, fatty acid
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