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An increase in bovine pyruvate carboxylase (PC; EC 6.4.1.1) at calving and during feed restriction corresponds with increased circulating nonesterified fatty acids as a consequence of negative energy balance. Regulation of PC mRNA and effect of specific combinations of saturated and unsaturated fatty acid profiles has yet to be explored. Our objective was to determine the effects of chain length, degree of saturation, and copresence of saturated and unsaturated fatty acids on activity of bovine PC promoter 1 (PCP1). For these experiments, Madin-Darby bovine kidney cells were transfected with a full-length bovine PCP1 construct from -1002 to +3 bp relative to the bovine PC gene transcription start site (bovine PCP1 (-1002_+3) ) ligated to a Firefly luciferase reporter, or with one of a series of nested 5' serial truncations (bovine PCP1 (-773_+3) , bovine PCP1 (-494_+3) , or bovine PCP1 (-222_+3) ). Cells were exposed for 23 h to either individual fatty acids (C16:0, C18:0, or C18:3n-3 cis) bound to BSA or to fatty acid mixtures in ratios of 90:10, 75:25, 50:50, or 25:75, corresponding to combinations of C16:0: C18:3n-3 cis or C18:0: C18:3n-3 cis. Total fatty acid concentration was 1.00 mM. Exposure to either C16:0 or C18:3n-3 cis alone elicited a significant increase in capacity to drive bovine PCP1 (-1002_+3) activity compared with 1% BSA in Dulbecco's Modified Eagle's Medium control treatment (2.29, 2.89, and 1.00 ± 0.26 fold of promoter induction for C16:0, C18:3n-3 cis, and control, respectively). Treatment with C18:3n-3 cis alone caused a greater increase in promoter activity compared with C16:0 alone, indicating a lesser response to C16:0 alone for bovine PCP1 (-1002_+3) . Interestingly, inclusion of C18:3n-3 cis, at any level of fatty acid ratios examined, in combination with C16:0 increased promoter activity of bovine PCP1 (-773_+3) or bovine PCP1 (-222_+3) compared with treatment with C16:0 alone or control. Data from the bovine PCP1 truncation and fatty acid copresence experiments reveal the potential for response elements of unsaturated fatty acids or fatty acid ligands in several bovine PCP1 promoter regions. In silico analysis of bovine PCP1 identified putative peroxisome proliferator-activated receptor α and sterol regulatory element binding protein binding sites which may be implicated in fatty acid signaling to alter bovine PCP1 activity. Pyruvate carboxylase promoter 1 activity that is mediated by unsaturated fatty acids acting through elements within -1002 and -222 bp of bovine PCPI may determine PC response during periods of negative energy balance in dairy cows., (Copyright © 2021 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Metabolic fates of fatty acids in tissue may be influenced by extracellular concentration and profile of fatty acids. Previous work has demonstrated the ability of C18:3n-3 cis to ameliorate the effects of C16:0- or C18:0-induced depression of pyruvate carboxylase (PC) mRNA expression. Pyruvate carboxylase catalyzes oxaloacetate synthesis and connects gluconeogenesis from lactate and fatty acid metabolism. Our objective was to determine the effects of co-presence of saturated and unsaturated fatty acids on cellular partitioning of [1- 14 C]C16:0 metabolism to CO 2 or acid-soluble products (ASP) in Madin-Darby bovine kidney cells and the role of PC in this relationship. We hypothesized that the ratio of saturated to unsaturated fatty acid pretreatments regulates [1- 14 C]C16:0 partitioning to CO 2 or ASP. Cells were exposed for 21 h to either individual fatty acids, C16:0, C18:0, C18:1n-9 cis, or C18:3n-3 cis, or to fatty acid combinations in 10:90, 25:75, 50:50, 75:25, or 90:10 ratios for 3 combinations: C16:0/C18:3n-3 cis, C18:0/C18:3n-3 cis, or C18:1n-9 cis/C18:3n-3 cis. Total fatty acid concentration was 1.0 mM during the 21-h pretreatment phase. Following the 21-h incubation phase with fatty acid combinations, cells were incubated in the presence of 1.0 mM [1- 14 C]C16:0 for 3 h to determine the rate of metabolism to CO 2 and ASP collection (per µg DNA -1 ·h -1 ). Pretreatment with either C16:0 or C18:0 alone significantly depressed subsequent oxidation of [1- 14 C]C16:0 to ASP by 62.7 and 41.2%, respectively, compared with C18:3n-3 cis pretreatment. Similar patterns were observed for [1- 14 C]C16:0 oxidation to CO 2 . Expression of PC mRNA was significantly decreased with exposure to either C16:0 or C18:0 compared with expression after exposure to either C18:3n-3 cis or control 1% BSA in Dulbecco's modified Eagle's medium. Expression of cytosolic phosphoenolpyruvate carboxykinase (PCK1) mRNA followed a similar pattern. Fatty acid treatments containing C18:1n-9 cis did not alter PC or PCK1 expression from control or C18:3n-3 cis results. Pearson coefficient correlations were determined for PC mRNA expression and rate of [1- 14 C]C16:0 metabolism to CO 2 or ASP, including ketones, and for PCK1 mRNA expression and rate of [1- 14 C]C16:0 metabolism to CO 2 or ASP. Production of CO 2 from [1- 14 C]C16:0 was positively correlated (r = 0.63) with PC expression, whereas ASP production from [1- 14 C]C16:0 only tended to positively correlate (r = 0.51) with PC mRNA expression. Production of CO 2 or ASP from [1- 14 C]C16:0 were both positively correlated (r = 0.80 and r = 0.69, respectively) with PCK1 expression. Results show a regulation of ketone production by Madin-Darby bovine kidney cells in response to saturated and unsaturated fatty acid pretreatments., (Copyright © 2020 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)