Modulating Oxaloacetate Flux in Dairy Cattle; At the Nexus of Energy and Glucose Metabolism

Linda M Beckett, Purdue University

Abstract

Oxaloacetate (OAA) is uniquely positioned in the tricarboxylic acid (TCA) cycle to be a substrate for the first committed step in gluconeogenesis or the oxidization of acetyl CoA. The anaplerotic action of pyruvate carboxylase (PC) synthesizes OAA, and the cataplerotic action of phosphoenolpyruvate carboxykinase (PEPCK) removes carbon from OAA to phosphoenolpyruvate (PEP), which is a gluconeogenic intermediate. Expression of pyruvate carboxylase and the cytosolic isoform of PEPCK, PCK1, are regulated by the addition of fatty acids in Madin-Darby bovine kidney (MDBK) cells. Previous studies using radioactive tracers demonstrated fatty acid induced changes in PC expression correlated with production of CO2 and acid soluble products (ASP). However, these previous data only captured full and partial oxidation of carbon and so specific changes in carbon flux that may occur through TCA cycle reactions were not identified. The studies conducted for this dissertation aimed to close the gap in knowledge regarding the relationship between fatty acids, PC, and PCK1/PCK2 expression and the flux of carbon through the OAA pool and TCA cycle and gluconeogenic reactions using heavy isotope tracer experiments. The central hypothesis wasthat the relative activities of PC and PEPCK control flux through the OAA pool and thus impact TCA cycle and gluconeogenic reactions. Thus, when PC expression increases, flux through the OAA pool and other TCA cycle and gluconeogenic reactions will increase, and the reverse will occur when PC expression decreases. The first study chapter describes studies that utilized PC knockdown and overexpression model to investigate the effect of changes in PC expression on flux of [U-13C] pyruvate. Overexpression of PC expression increased enrichment of TCA cycle intermediates and production of acid soluble products (ASP), indicating greater carbon flow through the TCA cycle and production of ketones. PC knockdown decreased enrichment of malate, aspartate, and fumarate which are products from the reversible reactions from the OAA pool, and increased ASP. Taken together, these data indicate an intermediate expression level of PC is needed to limit ketone production while maintaining TCA cycle oxidation. The study described in Chapter 3 investigated the effects of pretreating MDBK cells with varying ratios of palmitic acid and α-linolenic acid on PC expression and flux of [U-13C] pyruvate. PC expression was reduced due to the sole addition of palmitic acid, but an equimolar addition of palmitic acid to α-linolenic acid prevented the depression of PC expression, similar to control (no fatty acid addition) cells. Addition of only α-linolenic acid to cultures reduced enrichment of carbon from pyruvate into citrate, but increased enrichment of succinate with carbon from acetyl CoA, and there was increased oxidation of carbon from lactate to CO2 increased with increasing concentration of α-linolenic acid. PC expression negatively related to the enrichment of aspartate. These combined findings suggest carbon from pyruvate will be flowing towards citrate when PC expression is low, but α-linolenic acid increases PC expression and increases carbon flux and oxidation in the TCA cycle.

Degree

Ph.D.

Advisors

Casey, Purdue University.

Subject Area

Physiology|Neurosciences

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