DOI: 10.4231/v1f7-ga98
title: Fluxomics Combined with Shotgun Proteomics Reveals a Differential Response of Bovine Kidney Cells to Extracellular Palmitic and α-Linolenic Acid

description: &lt;p&gt;The data provided here are global proteome data generated when Madin Darby Bovine Kidney (MDBK) cells that were genetically or nutritionally modified. The MDBK cells were genetically modified by short hairpin RNA (shRNA) to knockdown pyruvate carboxylase (PC) expression or transduced with a lentiviral vector containing the tetracycline-on system to overexpress &lt;em&gt;PC&lt;/em&gt;. Then, a separate set of MDBK cells were incubated in different concentrations of palmitic acid and &alpha;-linolenic acid. These data pre presented and discussed in a manuscript submitted to Physiological Genomics.&lt;/p&gt;

&lt;p&gt;Previous data from our lab showed when MDBK cells were incubated in different combinations of palmitic acid and &alpha;-linolenic acid that &lt;em&gt;PC&lt;/em&gt; expression was reduced with higher additions of palmitic acid added to culture media. When palmitic acid was added in conjunction with &alpha;-linolenic acid, PC expression was prevented from being reduced. In addition, cells treated with palmitic acid had reduced full and partial oxidation of [1-&lt;sup&gt;14&lt;/sup&gt;C] palmitic acid and cell viability compared to control (no fatty acid addition) (Boesche and Donkin, 2020). Since PC is regulated by fatty acids, but is also a key carbon flux controlling enzyme, our objective of the current study was to characterize carbon flux through the tricarboxylic acid (TCA) cycle and measure the global proteome to create a map of metabolism when PC is genetically or nutritionally modified. MDBK cells were transduced with scramble control sequence shRNA not targeted for PC, shRNA targeted for PC, or a lentiviral vector with the tetracycline-on system to knockdown (PC-KD) or overexpress PC (PC-OE), respectively, and all cells were incubated in doxycycline for 48 hours. A separate set of MDBK cells were pretreated for 21 hours with different ratios of palmitic and &alpha;-linolenic acid with the following combinations: 1 mM palmitic acid to 0 mM &alpha;-linolenic acid (1P:0L), 0.75 mM palmitic acid to 0.25 mM &alpha;-linolenic acid (0.75P:0.25L), 0.50 mM palmitic acid to 0.5 mM &alpha;-linolenic acid (0.5P:0.5L). 0.25 mM palmitic acid to 0.75 mM &alpha;-linolenic acid (0.25P:0.75L), 0 mM palmitic acid to 1 mM &alpha;-linolenic acid (0P:1L), and vehicle control of no addition of fatty acids (BSA). All cells were incubated in [U-&lt;sup&gt;13&lt;/sup&gt;C] pyruvate for 2 hours and [1-&lt;sup&gt;14&lt;/sup&gt;C] palmitic acid and [U-&lt;sup&gt;14&lt;/sup&gt;C] lactate for 3 hours and flux measurements of the TCA cycle were collected, and protein from separate cell preparations were collected and global proteome analysis was conducted. Proteome analysis was only conducted on the Scramble, PC-KD, and PC-OE cells incubated in doxycycline and BSA, 1P:0L, 0.5P:0.5L, and 0P:1L pretreated cells.&lt;/p&gt;
description: Pyruvate carboxylase (PC) catalyzes the formation of the oxaloacetate a key TCA cycle intermediate and gluconeogenic substrate. Altering saturated to unsaturated fatty acid ratio alters PC expression, suggesting a central role in mediating carbon flow thr