Regulation of bovine pyruvate carboxylase gene promoters

Heather M White, Purdue University

Abstract

Pyruvate carboxylase (PC; EC 6.4.1.1) is critical in gluconeogenesis from lactate and maintenance of TCA cycle intermediates. The bovine PC gene contains three promoter sequences (P3, P2, and P1 from 5’ to 3’) that produce six 5’ untranslated region (UTR) variants. Products of P1 (5’ UTR A, B, C, and F) are glucogenic and lipogenic tissue specific while products of P2 and P3 (5’ UTR E and D, respectively) are expressed in several tissues. The central hypothesis of this dissertation is that changes in bovine PC promoter activity are responsible for changes in respective UTR variant expression and that physiological and environmental stressors regulate the bovine PC gene by altering promoter activity. The objectives of this work were to first develop a quantitative real-time RT-PCR multiplex assay for bovine PC 5’ UTR variants that would permit simultaneous characterization of variant expression patterns. Second, to utilize this multiplex assay to examine the variant profile during transition to lactation and feed restriction, and third, to characterize the mechanism of regulation of the bovine PC gene by fatty acids, PPARα agonist, glucocorticoids, and thermal stress. Development of a quantitative real-time RT-PCR multiplex assay for bovine PC 5’ UTR variants allowed for analysis of variant profiles in cows during transition to lactation and during feed restriction. These data indicated that increases in transcription rate and mRNA expression levels for bovine PC mRNA coding region and products of P1 are highly correlated and support the use of the multiplex assay described here, as a proxy measure of P1 activity. Furthermore, changes in PC mRNA expression at calving and during feed restriction were due to increases in expression of mRNA variant products of P1. Similarities in PC coding and variant mRNA responsiveness to transition to lactation and feed restriction indicate that regulation of the bovine PC gene during these periods is by the same mechanism, possibly mediated by changes coinciding with the onset of negative energy balance such as increased NEFA or glucocorticoid levels. Rat hepatoma cells were transiently transfected with promoter-luciferase contructs, containing bovine PC P1, P2, or P3, to quantify the direct effects to either stearic, oleic, or linoleic acids, dexamethasone, or a PPARα agonist on promoter activity. These data demonstrated a direct role of fatty acids, but not dexamethasone, to regulate PC expression. Fatty acids provided promoter specific regulation of PC promoters and was dependent on fatty acid and concentration. Promoter 1 was also upregulated by PPARα agonist, although P2 and P3 were not responsive. To examine if cells were responsive to circulating factors, including fatty acids, that are present in vivo, cells transfected with promoter-luciferase constructs containing P1 were incubated in the presence of serum from cows subjected to feed restriction or allowed ad libitum access to feed. These data indicate that P1 is activated by circulating factors found in serum of feed restricted cows. Promoter 1 responded similarly to serum from cows allowed ad libitum access to feed when fatty acids were added to match the NEFA concentration of serum from feed restricted cows. These data suggest a dominant role of NEFA concentrations and profiles in regulation of bovine PC P1. To elucidate the effect of fatty acid concentration and profile relevant to in vivo physiological conditions, fatty acids were combined in ratios designed to mimic the profile of plasma fatty acids during the period prior to calving, on the day of calving, on the day of calving in cows with induced fatty liver, and after calving. Fatty acid profile and concentration altered expression of key gluconeogenic enzymes, although the magnitude and directionality of the response was not uniform. Regulation of mRNA expression for these enzymes is likely part of the coordinated response of glucogenic tissues during transition to calving. (Abstract shortened by UMI.)

Degree

Ph.D.

Advisors

Donkin, Purdue University.

Subject Area

Animal sciences|Biochemistry|Physiology

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