Identification of promoter response elements that mediate propionate induction of bovine cytosolic phosphoenolpyruvate carboxykinase (PCK1) gene transcription.

Cytosolic phosphoenolpyruvate carboxykinase (PCK1) is a key enzyme for gluconeogenesis that is positively regulated by propionate in bovines at the transcription level. The specific elements that determine propionate responsiveness within the bovine PCK1 promoter are unknown. In silico promoter analysis of the bovine PCK1 gene revealed several clusters of transcription factor binding sites. In the present study, we determined the essentiality of the putative cyclic AMP response element (CRE) at -94 through -87 bp and the 2 putative hepatic nuclear factor 4α (HNF4α) binding elements at +68 through +72 and -1,078 through -1,074, respectively, in mediating bovine PCK1 promoter responses to propionate and other regulators, including butyrate, cyclic AMP (cAMP), and glucocorticoids. The wild-type bovine PCK1 promoter [PCK1(WT)] was ligated to a luciferase reporter gene and transfected into rat hepatoma (H4IIE) cells. Activities of PCK1(WT) were induced by approximately 2-, 2-, 4-, 8-, 9-, 18-, and 16-fold respectively when exposed to cAMP (as 1.0 mM 8-Br-cAMP), 5.0 µM dexamethasone, cAMP + dexamethasone, 2.5 mM propionate, cAMP + propionate, cAMP + dexamethasone + propionate, and 2.5 mM butyrate. Seven mutants lacking either one single site, 2 of the 3 sites, or all 3 sites, generated by site-directed mutagenesis, were tested. Responses to propionate and all other treatments were completely abolished when CRE at -94 through -87 bp and HNF4α at +68 through +72 bp were both deleted. Our data indicate that these 2 regulatory elements act synergistically to mediate the bovine PCK1 promoter responses to propionate as well as butyrate, cAMP, and dexamethasone. The activation of PCK1 through these regulatory elements serves to activate the metabolic potential of bovine toward gluconeogenesis when the primary substrate for gluconeogenesis, propionate, is also present.

Short communication: Regulation of hepatic gluconeogenic enzymes by dietary glycerol in transition dairy cows.

Nutritional status and glucose precursors are known regulators of gluconeogenic gene expression. Glycerol can replace corn in diets fed to dairy cows and use of glycerol is linked to increased rumen propionate production. The effect of dietary glycerol on the regulation of gluconeogenic enzymes is unknown. The objective of this study was to examine the effect of glycerol on expression of pyruvate carboxylase (PC), cytosolic and mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-C and PEPCK-M), and glucose-6-phosphatase. Twenty-six multiparous Holstein cows were fed either a control diet or a diet where high-moisture corn was replaced by glycerol from -28 through +56 d relative to calving (DRTC). Liver tissue was collected via percutaneous liver biopsy at -28, -14, +1, +14, +28, and +56 DRTC for RNA analysis. Expression of PC mRNA increased 6-fold at +1 and 4-fold at +14 DRTC relative to precalving levels. Dietary glycerol did not alter expression of PC mRNA expression. Expression of PEPCK-C increased 2.5-fold at +14 and 3-fold at +28 DRTC compared with +1 DRTC. Overall, dietary glycerol increased PEPCK-C expression compared with that of cows fed control diets. The ratio of PC to PEPCK-C was increased 6.3-fold at +1 DRTC compared with precalving and tended to be decreased in cows fed glycerol. We detected no effect of diet or DRTC on PEPCK-M or glucose-6-phosphatase mRNA, and there were no interactions of dietary treatment and DRTC for any transcript measured. Substituting corn with glycerol increased the expression of PEPCK-C mRNA during transition to lactation and suggests that dietary energy source alters hepatic expression. The observed increase in PEPCK-C expression with glycerol feeding may indicate regulation of hepatic gene expression by changes in rumen propionate production.

Regulation of bovine pyruvate carboxylase mRNA and promoter expression by thermal stress.

Pyruvate carboxylase (PC) catalyzes the rate-limiting step in gluconeogenesis from lactate and is a determinant of tricarboxylic acid cycle carbon flux. Bovine PC 5' untranslated region (UTR) mRNA variants are the products of a single PC gene containing 3 promoter regions (P3, P2, and P1, 5' to 3') that are responsive to physiological and nutritional stressors. The objective of this study was to determine the direct effects of thermal stress on PC mRNA and gene expression in bovine hepatocyte monolayer cultures, rat hepatoma (H4IIE) cells, and Madin-Darby bovine kidney epithelial (MDBK) cells. Hepatocytes were isolated from 3 Holstein bull calves and used to prepare monolayer cultures. Rat hepatoma cells and MDBK cells were obtained from American Type Culture Collection, Manassas, VA. Beginning 24 h after initial seeding, cells were subjected to either 37°C (control) or 42°C (thermal stress) for 24 h. Treatments were applied in triplicate in a minimum of 3 independent cell preparations. For bovine primary hepatocytes, endogenous expression of bovine PC mRNA increased (P < 0.1) with 24 h of thermal stress (1.31 vs. 2.79 ± 0.49, arbitrary units, control vs. thermal stress, respectively), but there was no change (P ≥ 0.1) in cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C) mRNA expression. Similarly, exposure of MDBK cells to thermal stress increased (P < 0.1) expression of bovine PC mRNA without altering (P ≥ 0.1) PEPCK-C mRNA expression. Conversely, there was no effect (P ≥ 0.1) of thermal stress on endogenous rat PC (0.47 vs. 0.30 ± 0.08, control vs. thermal stress) or PEPCK-C (1.61 vs. 1.20 ± 0.48, arbitrary units, control vs. thermal stress, respectively) mRNA expressions in H4IIE cells. To further investigate the regulation of PC, H4IIE cells were transiently transfected with bovine promoter-luciferase constructs containing either P1, P2, or P3, and exposed to thermal stress for 23 h. Activity of P1 was suppressed (P < 0.1) 5-fold, activity of P2 was unchanged (P ≥ 0.1), and activity of P3 was increased (P < 0.1) by 5.4-fold. These data indicate that response of bovine PC gene to thermal stress is through promoter regulation and suggest that there are unique characteristics of bovine PC promoters that may contribute to the physiological response to thermal stress.

Gluconeogenic enzymes are differentially regulated by fatty acid cocktails in Madin-Darby bovine kidney cells.

Expression of mRNA for pyruvate carboxylase (PC) is elevated at calving and during other physiological states when plasma NEFA concentrations are increased. The objective of this study was to determine the direct effects of fatty acids on expression of PC, cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C), mitochondrial PEPCK (PEPCK-M), and glucose-6-phosphatase (G6Pase) mRNA in Madin-Darby bovine kidney (MDBK) cells. Combinations of C14:0, C16:0, C18:0, C18:1n-6 cis, C18:2n-6 cis, and C18:3n-3 cis were created to mimic the profiles and concentrations in serum from far-off dry cows and late postcalving intervals (PRPT), the profile at calving (CALV), and the profile observed in cows induced to express fatty liver at calving (IFL). The MDBK cells were exposed to fatty acid mixtures for 24h at the following concentrations: 0.25 and 0.5mM for PRPT; 0.25, 0.5, and 1.0mM for CALV; and 0.5 and 1.0mM for IFL. Cells exposed to PRPT had greater PEPCK-C and tended to have greater G6Pase mRNA than control cells. Exposure of cells to 0.25mM PRPT increased expression of PEPCK-C compared with cells exposed to 0.5mM PRPT. Expression of PC and PEPCK-M did not differ with exposure to PRPT. Expression of PEPCK-C was decreased and that of PEPCK-M and G6Pase mRNA increased linearly in response to CALV. The ratio of PC:PEPCK-C mRNA was increased by the IFL mixture and in response to increasing amounts of the CALV fatty acid mixture. Treatment of cells with CALV or IFL increased the sum of PC 5' untranslated region (UTR) variants A, B, C, and F but did not alter PC 5' UTR D and E expression. The changes in PEPCK-C, G6Pase, and PC mRNA and the ratio of PC:PEPCK-C observed in MDBK cells in response to fatty acids suggests a role for fatty acid concentration and profile in mediating the expression of key gluconeogenic enzymes.

Differential regulation of bovine pyruvate carboxylase promoters by fatty acids and peroxisome proliferator-activated receptor-α agonist.

Pyruvate carboxylase (PC) is a critical enzyme in supplying carbon for gluconeogenesis and oxaloacetate for the tricarboxylic acid cycle. The bovine PC (EC 6.4.1.1) gene contains 3 promoter sequences (P3, P2, and P1 from 5' to 3'). Physiological stressors, including the onset of calving and feed restriction, lead to elevated nonesterified fatty acids and glucocorticoid levels that coincide with an increase in PC mRNA expression. The effects of elevated fatty acids on bovine PC mRNA expression and promoter function have not been determined. The objective of this experiment was to determine the direct effects of stearic, oleic, and linoleic acids, dexamethasone, and Wy14643 (a peroxisome proliferator-activated receptor-α agonist) on bovine PC promoter activity. Promoter-luciferase constructs, containing 1,005 bp of P1, 1,079 bp of P2, or 1,010 bp of P3, were transiently transfected into rat hepatoma (H4IIE) cells. Cells were then treated with 1mM stearic, oleic, or linoleic acids, 1 µM dexamethasone, or 10 µM Wy14643 for 23 h. Activity of P1 was suppressed with exposure to stearic acid (1.58 vs. 6.19±0.81 arbitrary units for stearic vs. control, respectively) and enhanced with exposure to Wy14643 (9.26 vs. 6.19±0.81 arbitrary units for Wy14643 vs. control, respectively). Conversely, stearic acid enhanced P3 activity (2.55 vs. 0.40±0.33 arbitrary units for stearic vs. control, respectively). Dexamethasone, linoleic acid, and oleic acid failed to elicit a response from any of the promoters tested. These data demonstrate the direct role of fatty acids in regulating PC expression and indicate that fatty acids provide promoter-specific regulation of PC promoters.

Characterization of bovine pyruvate carboxylase promoter 1 responsiveness to serum from control and feed-restricted cows.

Pyruvate carboxylase (PC; EC 6.4.1.1) is critical in gluconeogenesis from lactate and maintenance of tricarboxylic acid cycle intermediates. Whereas increases in PC mRNA have been observed during feed restriction, the mechanism of regulation is unknown; however, coinciding increases in circulating NEFA concentrations suggests that fatty acids may contribute to regulation of gene expression during feed restriction. The objective of this study was to examine the direct effect of exposure to serum from full-fed control cows with serum from cows that were restricted to 50% of ad libitum intake for 5 d on PC expression in vitro. Rat hepatoma (H4IIE) cells were transiently transfected with bovine promoter-luciferase constructs containing bovine PC promoter 1 and treated with serum from control cows, serum from feed-restricted cows, or modified serum. Modified serum pools were generated by supplemented serum from control cows with C14:0, C16:0, C18:0, C18:1n-9 cis, C18:2n-6 cis, and C18:3n-3 cis to match the total NEFA in serum from feed-restricted cows (1.3 mM) in the relative proportion found in serum from control or feed-restricted cows. Exposure of cells to serum from feed-restricted cows increased (P < 0.05) PC promoter 1 activity 2.2-fold compared with cells exposed to control cow serum. Exposure to serum from control cows with fatty acids added to a NEFA concentration of 1.3 mM to reflect the fatty acid profile of control and feed-restricted cows increased (P < 0.05) promoter 1 activity 2.1- and 2.5-fold, respectively, compared with cells incubated with control cow serum. There was no difference (P ≥ 0.05) in promoter 1 activity in cells treated with modified serum compared with serum from feed-restricted cows. These data indicate that promoter 1 is activated by fatty acids found in serum of feed-restricted cows. These data suggest a role of NEFA to regulate expression of bovine PC mRNA through specific activation of PC promoter 1.

Bovine pyruvate carboxylase 5' untranslated region variant expression during transition to lactation and feed restriction in dairy cows.

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.

Feeding value of glycerol as a replacement for corn grain in rations fed to lactating dairy cows.

Growth of the corn ethanol industry has created a need for alternatives to corn for lactating dairy cows. Concurrent expansion in soydiesel production is expected to increase availability and promote favorable pricing for glycerol, a primary co-product material. The objective of this study was to determine the feeding value of glycerol as a replacement for corn in diets fed to lactating dairy cattle. Sixty lactating Holstein cows housed in individual tie stalls were fed a base diet consisting of corn silage, legume forages, corn grain, soyhulls, roasted soybeans, and protein supplements. After a 2-wk acclimation period, cows were fed diets containing 0, 5, 10, or 15% refined glycerol for 56 d. Cows were milked twice daily and weekly milk samples were collected. Milk production was 36.3, 37.2, 37.9, and 36.2 +/- 1.6 kg/d and feed intake was 23.8, 24.6, 24.8, and 24.0 +/- 0.7 kg/d for 0, 5, 10, and 15% glycerol treatments, respectively, and did not differ except for a modest reduction in feed intake during the first 7 d of the trial for 15% glycerol (treatment x time effect). Milk composition was not altered by glycerol feeding except that milk urea nitrogen was decreased from 12.5 +/- 0.4 to 10.2 +/- 0.4 mg/dL with glycerol addition. Cows fed diets containing 10 and 15% glycerol gained more weight than those fed rations containing 0 or 5% glycerol but body condition scores did not differ with glycerol feeding. The data indicate that glycerol is a suitable replacement for corn grain in diets for lactating dairy cattle and that it may be included in rations to a level of at least 15% of dry matter without adverse effects on milk production or milk composition.

Feeding conjugated linoleic acid partially recovers carcass quality in pigs fed dried corn distillers grains with solubles.

Dried corn distillers grains with solubles (DDGS) fed to swine may adversely affect carcass quality due to the high concentration of unsaturated fat. Feeding CLA enhances pork quality when unsaturated fat is contained in the diet. The effects of CLA on growth and pork quality were evaluated in pigs fed DDGS. Diets containing 0, 20, or 40% DDGS were fed to pigs beginning 30 d before slaughter. At 10 d before slaughter, one-half of each DDGS treatment group was fed 0.6% CLA or 1% choice white grease. Carcass data, liver- and backfat-samples were collected at slaughter. Longissimus muscle area, 10th-rib back-fat depth, last rib midline backfat depth, LM color, marbling, firmness and drip loss, and bacon collagen content were not altered by DDGS or CLA. Outer layer backfat iodine values were increased (P 0.05) for pigs fed DDGS. Feeding CLA decreased (P

Translational efficiency of bovine pyruvate carboxylase 5' untranslated region messenger ribonucleic acid variants.

The bovine pyruvate carboxylase (PC) gene is expressed as 6 alternatively spliced variants that share a common open reading frame but that differ within their 5' untranslated regions (UTR). The PC 5' UTR variants (A through F) contain 6 combinations of 5 exons and are 68, 253, 363, 89, 226, 178 bp in length, respectively. The objective of this experiment was to determine whether or not the bovine PC mRNA variants exhibit different translational efficiencies. Each bovine PC 5' UTR variant was linked to the firefly luciferase coding region, and the resulting constructs were transcribed and translated in a rabbit reticulocyte lysate assay. All constructs resulted in synthesis of luciferase protein. The abundance of luciferase protein synthesized from the UTR of bovine PC 5' D was greater (P < 0.05) than synthesis from either PC 5' UTR C or E, and the abilities of UTR D, A, B, and F to drive protein translation were similar. The disproportionate contribution to protein synthesis of the PC 5' D UTR compared with UTR variant C or E indicates a complexity of control for PC enzyme synthesis in the bovine that is dependent on the profile of PC variants. These observations are consistent with differences in PC variant expression that have been observed in vivo and indicate that when PC mRNA is elevated, the pattern of variants directs an increase in PC activity through augmented PC enzyme synthesis.