Author ORCID Identifier

https://orcid.org/0000-0003-2923-2158

Date Available

11-13-2017

Year of Publication

2017

Degree Name

Doctor of Philosophy (PhD)

Document Type

Doctoral Dissertation

College

Agriculture, Food and Environment

Department/School/Program

Animal and Food Sciences

First Advisor

Dr. James C. Matthews

Abstract

Improvement of feeding regimens for production animals has been hindered by a lack of fundamental knowledge about how the capacity to regulate nutrient absorption across cell membranes affects the function of nutrient metabolizing enzymes. The objective is to determine if the activities and protein content of system X-AG glutamate transporter, its regulatory protein (GTRAP3-18 and ARL6IP1), glutamine synthetase (GS) and glutathione (GSH) content, changes in liver (Experiment 1), longissimus dorsi (LM) and subcutaneous adipose tissue (SF) (Experiment 2) as beef steers transitioned from predominantly-lean (growing) to -lipid (finished) tissue accretion phases. In liver (Experiment 1), system X-AG activity in canalicular membranes was abolished as steers developed from growing to finished stages but did not change in basolateral membranes. EAAC1 protein content in liver homogenates decreased in finished vs. growing steers, whereas GTRAP3-18 and ARL6IP1 content increased and GLT-1 content did not change. Concomitantly, hepatic GS activity decreased in finished steers whereas GS protein content did not differ. Hepatic GSH content did not differ in finished vs. growing steers. These results demonstrate a negative functional relationship between GTRAP3-18 and system X-AG activity with glutamine synthesis capacity in livers of fattened cattle. In addition to liver, skeletal muscle and adipose tissues play important roles in maintaining whole-body glutamate and nitrogen homeostasis. In Experiment 2, Western blot analysis of LM homogenates showed decreased EAAC1 and GS content, whereas GTRAP3-18 and ARL6IP1 did not differ in finished vs. growing steers. GSH content in LM was increased in finished vs. growing steers in concomitance with increased mRNA expression of GSH-synthesizing enzymes. In SF, GTRAP3-18 and ARL6IP1 content was increased, whereas EAAC1 and GS content did not differ. Concomitantly, GSH content in SF was decreased in finished vs. growing steers in parallel with decreased mRNA expression of GSH-metabolizing enzymes. These results demonstrate that the negative regulatory relationship between GTRAP3-18 and ARL6IP1 with EAAC1 and GS expression, which exists in liver, does not exist in LM and SF of fattened cattle; and antioxidant capacity in LM and SF changes and differs as steer compositional gain shifts from lean to lipid phenotype. To further explore the upstream regulatory machinery of EAAC1, transcriptome analysis (Experiment 3) was conducted to gain a greater understanding of hepatic metabolic shifts associated with the change in whole-body compositional gain of growing vs. finished beef steers. The expression of upstream regulators of EAAC1 was decreased in a manner consistent with the decreased EAAC1 activity in Experiment 1. Bioinformatic analysis found that, for amino acid metabolism, finished steers had increased capacities for ammonia, arginine, and urea production, and shunting of amino acid carbons into pyruvate. For carbohydrate metabolism, capacity for glycolysis was inhibited, whereas glycogen synthesis was stimulated in finished steers. For lipid metabolism, finished steers showed decreased capacity for fatty acid activation and desaturation, but increased capacity for fatty acid b-oxidation and lipid storage. In addition, redox capacity and inflammatory responses were decreased in finished steers. Collectively, these data describe novel regulatory relationships of system X-AG in liver and peripheral tissues, and the metabolic mechanisms that control nutrient use efficiency, as beef steers develop from lean to lipid phenotypes.

Digital Object Identifier (DOI)

https://doi.org/10.13023/ETD.2017.492

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