Date of Award

Spring 2015

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Animal Science

First Advisor

Shawn S. Donkin

Committee Chair

Shawn S. Donkin

Committee Member 1

Kolapo Ajuwon

Committee Member 2

Dorothy Teegarden

Committee Member 3

Jonathan R. Townsend

Abstract

Phosphoenolpyruvate carboxykinase (PCK, GTP; EC4.1.1.32) catalyzes the conversion of oxaloacetate (OAA) to phosphoenolpyruvate (PEP), a critical reaction for gluconeogenesis. The two isoforms of PCK, namely cytosolic PCK (PCK1), and the mitochondrial PCK (PCK2) are abundantly expressed in liver and kidney. Increased PCK1 activity and mRNA expression in liver is observed as feed intake recovers during early lactation in dairy cows, a condition linked to increased ruminal propionate production. The central hypothesis of this dissertation is that propionate regulates its own metabolism in liver of dairy cattle through regulation of the PCK1 gene. The objectives of this dissertation were to determine: 1) the effect of elevated propionate supply on expression of key genes for gluconeogenesis, particularly PCK1, in liver of dairy cattle; 2) the effect of propionate on expression of PCK1 mRNA under controlled hormonal status in cultured primary bovine hepatocytes; 3) the effect of propionate on the transcriptional activity of the bovine PCK1 promoter; 4) the molecular mechanism and cis-elements within the bovine PCK1 promoter responsible for the induction of PCK1 expression by propionate.^ Postruminal infusion of propionate to mid-lactation cows and intravenous infusion of propionate to neonatal calves allowed evaluation of the effects of increased propionate supply on hepatic expression of the gluconeogenic genes. These data indicated that mRNA expression of PCK1 and G6PC were at least maintained by propionate infusion in mid-lactation cows despite the greatly elevated blood insulin levels that suppressed the expression of these two genes without propionate infusion. When blood insulin levels were not elevated, as observed in the neonatal calves, the mRNA expression of PCK1 and G6PC was increased.^ In vitro culture of bovine hepatocytes allowed examination of the direct effect of propionate on expression of PCK1 mRNA independent of hormonal status. In this study, bovine hepatocytes were isolated from neonatal calves and cultured with propionate, insulin, cAMP, dexamethasone or their combinations. These data demonstrated a direct role of propionate to induce PCK1 mRNA expression in the bovine hepatocytes. Propionate-induced PCK1 mRNA expression was mimicked by cAMP, but not by dexamethasone, and these effects were inhibited by addition of insulin.^ To investigate whether propionate regulates the PCK1 expression at transcriptional level, the promoter of the bovine PCK1 gene was cloned and ligated to Firefly luciferase reporter and transiently transfected into the rat hepatoma H4IIE cells. The effects of acetate, propionate, butyrate, glycerol, cAMP, dexamethasone and insulin on the activity of PCK1 promoter were determined. These data indicated that the induction of PCK1 mRNA expression by propionate was through direct promoter activation. The induction of bovine PCK1 promoter by propionate or butyrate was not a general effect of short-chain fatty acids (SCFA) as acetate was without effect. Additionally, bovine PCK1 promoter was stimulated by cAMP and dexamethasone, however, to a much less extent compared with propionate. Propionate, cAMP, and dexamethasone act synergistically to induce PCK1 transcription. Addition of insulin repressed the promoter activation stimulated by cAMP and dexamethasone, but failed to inhibit the propionate-induced promoter activation.^ Furthermore, the underlying molecular mechanism and cis-regulatory elements within the bovine PCK1 promoter that is responsible for the responses to propionate, butyrate, cAMP and dexamethasone, as well as the synergistic effect of propionate, cAMP, and dexamethasone were investigated. Particularly, the role of cAMP response element (CRE) and the binding sites for Hepatic Nuclear Factor 4&agr; (HNF4&agr;) were determined by site-direct mutagenesis. These data suggested that propionate, butyrate, cAMP and dexamethasone shared common signaling pathway to mediate bovine PCK1 transcription, and the consensus sequences for CRE located at -94 through -87 bp and HNF4&agr; site located at +68 through +72 bp relative to the transcription start site of the bovine PCK1 gene were responsible for the maximum promoter induction of PCK1 promoter as double deletions of these two sites completely abolished the responses to propionate, butyrate, cAMP, dexamethasone as well as the synergistic induction by propionate, cAMP and dexamethasone.^ Together, these data suggest a feed-forward mechanism that links propionate, a primary SCFA produced in the rumen, with its own metabolic fate through regulation the transcription of PCK1, the key flux-regulating enzyme for gluconeogenesis.

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