Studies on the role of expression of phosphoenolpyruvate carboxykinase gene in the development of bovine lactation ketosis

Abstract

<p>Changes in expression of the phosphoenolpyruvate carboxykinase (PEPCK) gene, which codes for a key regulatory enzyme of gluconeogenesis, were studied in bovine liver tissue. Induction of ketosis was attempted in cows by a protocol of restricting feed intake (FR) and supplementing with 1,3-butanediol (BD), a ketogenic precursor, beginning at 15 d postpartum (FRBD protocol). Twenty Holstein cows were distributed in equal numbers among four treatments: normal control, obese control, normal cows given FRBD, and obese cows given FRBD. Liver biopsies were obtained at nine different times during early lactation: 10 d prepartum, 5 and 14 d postpartum (just before FRBD begins), 21, 28, and 36 d postpartum (about the time when ketosis should begin), and 49 and 63 d postpartum. Not all FRBD cows developed clinical ketosis; some only had subclinical ketosis;Relative concentrations of mRNA for phosphoenolpyruvate carboxykinase (PEPCK) in the lactating cows increased up to about d 35 postpartum, then a dramatic decrease occurred around d 42 postpartum, the time when clinical signs of ketosis became evident. The control groups did not exhibit as dramatic a decrease (P 0.05) with PEPCK mRNA (-0.20) and with concentrations of protein (-0.18), suggesting that both PEPCK mRNA and PEPCK protein decreased as the triacylglycerol to glycogen ratio increased;To amplify the cytosolic PEPCK gene in bovine liver, primers were synthesized from selected homologous sequences of the rat PEPCK gene for amplification by the polymerase chain reaction (PCR). A 200-base pair (bp) fragment was amplified from bovine genomic DNA, whereas a 960-bp fragment was amplified from bovine cDNA. The amplified bovine PEPCK cDNA was found to be 80% homologous to the rat PEPCK cDNA.</p>