Fluxomics combined with shotgun proteomics reveals a differential response of bovine kidney cells to extracellular palmitic and α-linolenic acid

Pyruvate carboxylase (PC) catalyzes the formation of oxaloacetate, a TCA cycle intermediate and gluconeogenic substrate. Altering saturated to unsaturated fatty acid ratio alters <i>PC</i> expression, suggesting a central role in mediating carbon flow through metabolic pathways. Herein, we describe changes in metabolic flux of TCA cycle intermediates and proteome in Madin-Darby bovine kidney (MDBK) cells with <i>PC</i> expression knocked-down (PC-KD), overexpressed (PC-OE), unaltered using a Scramble control, or cells pretreated for 21 h with vehicle control bovine serum albumin (BSA) or different ratios of palmitic acid (P) and α-linolenic acid (L) ranging from 1 mM P:0 mM L (1P:0L) to 0P:1L. All cells were collected for proteome analysis and to measure [U-¹³C] pyruvate flux or oxidation of [1-¹⁴C] palmitic acid and [U-¹⁴C] lactate. Compared with Scramble, ¹³C enrichment of all TCA cycle intermediates was greater in PC-OE, but all were reduced in PC-KD except succinate. Proteins greater in abundance in both cell lines included solute transporters, propionyl CoA carboxylase, and fatty acid binding protein 3. Relative to BSA, 1P:0L increased cell death and increased ¹³C flux to citrate but decreased enrichment of succinate. Abundance of citrate synthase, aconitase, glutamine aminotransferases, and succinyl CoA synthetases was greater in 1P:0L, but not different in other pretreatments. Results indicate preferential utilization of pyruvate and amino acids by 1P:0L cells whereas 0P:1L treated cells show preference for α-linolenic acid metabolism. <i>PC</i> regulates metabolic flux, C18:3<i>n</i> - 3 <i>cis</i> prevents lipotoxicity, and both alterations in <i>PC</i> and the addition of C18:3<i>n</i> - 3 <i>cis</i> promote oxidation of fatty acids.<b>NEW & NOTEWORTHY</b> <i>PC</i> overexpression increases the capacity for fatty acid oxidation, whereas <i>PC</i> knockdown requires extracellular amino acids to support TCA cycle intermediates. Cells incubated in palmitic acid demonstrated dependency of pyruvate and amino acids as substrates for the TCA cycle. Exposure to α-linolenic acid reduces the dependency of pyruvate as a substrate likely because carbon from α-linolenic acid can be used to supply TCA cycle intermediates whereas palmitic acid carbon is not used.