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

Abstract

Pyruvate carboxylase (PC) catalyzes the formation of oxaloacetate, a TCA cycle intermediate and gluconeogenic substrate. Altering saturated to unsaturated fatty acid ratio alters PC 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 PC 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. PC regulates metabolic flux, C18:3n - 3 cis prevents lipotoxicity, and both alterations in PC and the addition of C18:3n - 3 cis promote oxidation of fatty acids.NEW & NOTEWORTHY PC overexpression increases the capacity for fatty acid oxidation, whereas PC 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.

Keywords: MDBK cells; metabolic flux; palmitic acid; pyruvate carboxylase; α-linolenic acid.