Control of bovine pyruvate carboxylase expression by saturated and unsaturated fatty acids and impact on fatty acid metabolism
Pyruvate carboxylase (PC; EC 126.96.36.199) ostensibly links carbohydrate and lipid metabolism by supplying oxaloacetate (OAA) from the carboxylation of pyruvate. The OAA pool is necessary in maintaining the anaplerotic supply of carbons to the TCA cycle, and to promote the complete oxidation, to CO 2, of acetyl-CoA. The actions of PC in supplying OAA are critical during periods of increased cellular lipid load, including the negative energy balance experienced by periparturient dairy cows. The bovine PC gene contains three promoter sequences, with products of the proximal promoter (PCP1) being specific to glucogenic and lipogenic tissues. Previous work links control of bovine PC mRNA expression to nonesterified fatty acids (NEFA), but the direct effects of saturated and unsaturated fatty acids on PC mRNA expression and subsequent fatty acid metabolism were unclear. The central hypothesis of this dissertation is that the ratio of the most prevalent circulating saturated and unsaturated fatty acids in dairy cows regulates PC flux and subsequent fatty acid metabolism. The objectives of this dissertation were, first, to evaluate the response of PC mRNA to the copresence of the most abundant circulating saturated and unsaturated fatty acids in periparturient dairy cows. Second, to determine the direct effects of these saturated and unsaturated fatty acids, and their combinations, on the cellular oxidation of fatty acids, and the potential relationship to PC, and third, to examine the effects of the copresence of saturated and unsaturated fatty acids on the transcriptional activity of bovine PCP1. Bovine PC mRNA expression was first examined, in response to copresence of saturated and unsaturated fatty acids, in a bovine kidney cell line and in bovine primary hepatocytes. Both C16:0 or C18:0 alone significantly depressed PC mRNA in bovine kidney cells. Inclusion of unsaturated C18:3n-3 cis restored PC expression to levels similar to control in bovine kidney cells. Data from bovine primary hepatocytes displayed a similar numerical pattern in response to fatty acid combinations, although means did not differ. However, incubation with C18:3n-3 cis alone resulted in nearly two-fold increase in PC mRNA expression, compared to PC mRNA expression after control or saturated fatty acid exposure. Treatment with monounsaturated C18:1n-9 cis did not alter PC mRNA expression in either cell model. Results indicate that C18:3n-3 cis is a potent alleviator of saturated fatty acid-induced depression of PC mRNA expression. To determine the impact of alterations in PC mRNA expression on cellular metabolism of fatty acids, the flux of radiolabeled C16:0 to CO2 or to acid-soluble metabolites, including ketone bodies, was analyzed after pretreatment of bovine kidney cells with combinations of saturated and unsaturated fatty acids which are most prevalent in periparturient dairy cows. Prior exposure to either C16:0 or C18:0 alone significantly depressed both complete and partial oxidation compared to exposure to C18:3n-3 alone. Data for complete oxidation of radiolabeled C16:0 to CO2 were correlated (r = 0.63; P < 0.05) with PC mRNA expression, suggesting a potential relationship between PC flux and subsequent fatty acid metabolism. Oxidation of radiolabeled C16:0 to CO2 was also correlated (r = 0.80; P < 0.05) to expression of cytosolic phosphoenolpyruvate carboxykinase (PCK1). The data indicate that although PC is regulated by fatty acids and in turn modulates the oxidation of fatty acids, the concomitant increase in PCK1 may oppose these effects by pulling carbon from the OAA pool toward gluconeogenesis. Factors remain to be identified that permit increased PC flux while maintaining, but not enhancing, PCK1 flux. The role of bovine PCP1 in regulating the expression of PC mRNA in response to the copresence of saturated and unsaturated fatty acids prevalent in periparturient dairy cows was evaluated using bovine PCP1 ligated to a Firefly luciferase reporter and transiently transfected into bovine kidney cells. Bovine PCP1 activity increased in response to either C16:0 or C18:3n-3 cis, and combinations of those fatty acids, indicating a dominant role of C18:3n-3 cis in controlling bovine PCP1 activity. Truncations of bovine PCP1 indicate a region responsive to fatty acids located between -773 to -494 bp (PCP1(-773_+3)) relative to transcription start site (TSS), as activity in response to fatty acid greatly diminished with truncation of this region. In silico analysis of bovine PCP1 indicates several transcription factor binding site consensus sequences, including peroxisome proliferator activated receptor (PPAR) α and sterol regulatory element binding proteins (SREBP) sites, that are candidate response elements for control by C18:3n-3 cis and C16:0 or C18:0. Results indicate a dominant role of C18:3n-3 cis to signal through bovine PCP1(-773_+3) containing a PPARα binding site. A SREBP binding site within bovine PCP1(-773_+3) is a candidate for saturated fatty acid responsiveness within this promoter region. Together, these data indicate control by saturated and unsaturated fatty acids prevalent in periparturient bovine on PC mRNA expression, regulated through the PC proximal promoter, and in determining subsequent cellular fatty acid metabolism. Responsiveness of bovine PCP1 to saturated and unsaturated fatty acids may be mediated through identified putative binding sites. The cellular oxidative capacity, dependent upon the pool of OAA generated by PC, can help determine the response to physiological factors, including fatty acids, which increase during metabolically stressful periods in dairy cows.
Donkin, Purdue University.
Cellular biology|Animal sciences|Physiology
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