Bovine pyruvate carboxylase 5′ untranslated region variant expression during transition to lactation and feed restriction in dairy cows1

Abstract

Pyruvate carboxylase (PC; EC 6.4.1.1) is a critical enzyme for gluconeogenesis and maintenance of tricarboxylic acid (TCA) cycle intermediates, and expression of PC mRNA is increased at calving and during feed restriction. The bovine PC gene contains 3 promoters (3, 2, and 1 from 5' to 3') that produce 6 mRNA 5' variants (A through F). Products of promoter 1, untranslated region (UTR) variants A, B, C, and F are specifically expressed in glucogenic and lipogenic tissues. The objective of this study was to develop a quantitative PCR-based assay for bovine PC 5' UTR variants that would permit simultaneous characterization of PC variant expression and to determine the pattern of PC variant expression during the transition to lactation and during feed restriction. Primer combinations specific to the coding region of PC and the 5' UTR for variants D, E, and F were used with Taqman probes in a real-time PCR multiplex assay to simultaneously determine total PC mRNA and expression of each UTR variant. The intraassay and interassay CV were less than 2 and 10%, respectively. Total PC mRNA and PC 5' UTR variant profile was determined for liver biopsy samples collected from Holstein cows (n = 8) at -28, +1, and +28 d relative to calving (DRTC) and from mid-lactation Holstein cows subjected to either feed restriction (n = 8) or fed for ad libitum intake (n = 8). The expression of PC mRNA corresponding to the coding region of PC and PC 5' UTR variant regions A, B, C, and F increased (P < 0.05) 4-fold with feed restriction and 6-fold at calving. Nuclei isolated from liver biopsy samples and used to determine the rates of PC gene transcription indicate changes in the abundance of PC 5' mRNA variants A, B, C, and F that are due to corresponding changes in the rate of transcription of the bovine PC gene. The data support the use of the multiplex assay described here as a proxy measure of the activity of bovine PC promoter 1. The increased PC mRNA expression observed at calving and during feed restriction is the result of specific increases in 5' UTR variants A, B, C, and F due to increased transcriptional activity of promoter 1 of the PC gene.

Periparturient dairy cows do not exhibit hepatic insulin resistance, yet adipose-specific insulin resistance occurs in cows prone to high weight loss

“…Moreover, a negative correlation was observed between peripartum basal plasma insulin concentrations and the relative expression of PC (r = −0.85, P < 0.0001). These findings are in agreement with others (Graber et al, 2010;White et al, 2011), as the rate of hepatic gluconeogenesis must abruptly double during the peripartum period to meet the high glucose demand of the mammary gland (Bell and Bauman, 1997).…”

Hepatic patatin-like phospholipase domain-containing protein 3 sequence, single nucleotide polymorphism presence, protein confirmation, and responsiveness to energy balance in dairy cows

“…Given the regulation of hepatic PNPLA3 by FA in rodents and human hepatocytes (Huang et al, 2010), it is possible that bovine PNPLA3 is also responsive to FA, which are known to be dynamic during changes in energy balance in ruminant animals. Circulating NEFA concentrations were increased in both the feedrestricted cows (White et al, 2011a) and during +1 and +14 DRTC (Carvalho et al, 2011), as reported previously. Expression of PNPLA3 mRNA in each animal was negatively correlated with plasma NEFA concentration, with a modest, although significant rvalue of −0.33.…”

“…Liver biopsy samples from feed-restricted and transition cows were collected during experiments conducted at Purdue University (West Lafayette, IN). Full details regarding experimental design, methods, animal performance, and plasma NEFA have been reported previously (Carvalho et al, 2011;White et al, 2011a). Liver biopsy samples collected at −14, +1, +14, +28 d relative to calving (DRTC) in control-fed transition cows, and in mid-lactation cows after 5 d of ad libitum access to feed or 50% feed restriction were collected for isolation of RNA.…”

“…Abundance of PNPLA3 mRNA was determined by real-time quantitative PCR and normalized to 18S as described previously (White et al, 2011a(White et al, , 2012 in samples collected from feed-restricted and transition cows. Primers were as follows: PNPLA3: AGACAT-GCCCGGCGACATCCA (forward) and CAGCAGG-TACACTGGGAC CTCTGA (reverse); 18S (Bos taurus gene number 493779): ACCCATTCGAACGTCTG CCCTATT (forward) and TCCTTGGATTGTGG-TAGCCGTTTCT (reverse).…”

The Role of TCA Cycle Anaplerosis in Ketosis and Fatty Liver in Periparturient Dairy Cows

“…Examination of the balance of PC and PEPCK during different physiological states or during nutritional challenges highlights the importance of anaplerotic balance. Increased hepatic expression of PC, but not PEPCK, is observed at calving [ 34 , 35 , 36 , 37 ] and may allow for increased oxidative capacity within the mitochondria. Regulation of PC during the transition to lactation is through regulation of PC Promoter 1, which is upregulated by fatty acids that mimic those in circulation at calving [ 32 , 35 , 38 , 39 ].…”