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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.

Cytosolic phosphoenolpyruvate carboxykinase (PCK1) is a critical enzyme within the metabolic networks for gluconeogenesis, hepatic energy metabolism, and tricarboxylic acid cycle function, and is controlled by several transcription factors including hepatic nuclear factor 4α (HNF4α). The primary objective of the present study was to determine whether propionate regulates bovine PCK1 transcription. The second objective was to determine the action of cyclic AMP (cAMP), glucocorticoids, and insulin, hormonal cues known to modulate glucose metabolism, on bovine PCK1 transcriptional activity. The proximal promoter of the bovine PCK1 gene was ligated to a Firefly luciferase reporter and transfected into H4IIE hepatoma cells. Cells were exposed to treatments for 23 h and luciferase activity was determined in cell lysates. Activity of the PCK1 promoter was linearly induced by propionate, and maximally increased 7-fold with 2.5 mM propionate, which was not muted by 100 nM insulin. Activity of the PCK1 promoter was increased 1-fold by either 1.0 mM cAMP or 5.0µM dexamethasone, and 2.2-fold by their combination. Induction by cAMP and dexamethasone was repressed 50% by 100 nM insulin. Propionate, cAMP, and dexamethasone acted synergistically to induce the PCK1 promoter activity. Propionate-responsive regions, identified by 5' deletion analysis, were located between -1,238 and -409 bp and between -85 and +221 bp. Deletions of the core sequences of the 2 putative HNF4α sites decreased the responsiveness to propionate by approximately 40%. These data indicate that propionate regulates its own metabolism through transcriptional stimulation of the bovine PCK1 gene. This induction is mediated, in part, by the 2 putative HNF4α binding sites in the bovine PCK1 promoter.

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.

Elevated needs for glucose in lactating dairy cows are met through a combination of increased capacity for gluconeogenesis and increased supply of gluconeogenic precursors, primarily propionate. This study evaluated the effects of propionate on mRNA expression of cytosolic phosphoenolpyruvate carboxykinase (PCK1), mitochondrial phosphoenolpyruvate carboxykinase (PCK2), pyruvate carboxylase (PC), and glucose-6-phosphatase (G6PC), key gluconeogenic enzymes, and capacity for glucose synthesis in liver of dairy cattle. In experiment 1, six multiparous mid-lactation Holstein cows were used in a replicated 3×3 Latin square design consisting of a 6-d acclimation or washout phase followed by 8h of postruminal infusion of either propionate (1.68mol), glucose (0.84mol), or an equal volume (10mL/min) of water. In experiment 2, twelve male Holstein calves [39±4 kg initial body weight (BW)] were blocked by birth date and assigned to receive, at 7d of age, either propionate [2mmol·h(-1)·(BW(0.75))(-1)], acetate [3.5mmol·h(-1)·(BW(.75))(-1)], or an equal volume (4mL/min) of saline. In both experiments, blood samples were collected at 0, 2, 4, 6, and 8h relative to the start of infusion and liver biopsy samples were collected at the end of the infusion for mRNA analysis. Liver explants from experiment 1 were used to measure tricarboxylic acid cycle flux and gluconeogenesis using (13)C mass isotopomer distribution analysis from (13)C3 propionate. Dry matter intake and milk yield were not altered by infusions in cows. Serum insulin concentration in cows receiving propionate was elevated than cows receiving water, but was not different from cows receiving glucose. Hepatic expression of PCK1 and G6PC mRNA and glucose production in cows receiving propionate were not different from cows receiving water, but tended to be higher compared with cows receiving glucose. Hepatic expression of PCK2 and PC mRNA was not altered by propionate infusion in cows. Blood glucose, insulin, and glucagon in calves receiving propionate were not different than controls. Calves receiving propionate had increased PCK1 mRNA, tended to have increased G6PC mRNA, and had similar PC mRNA compared with saline controls. These data indicate a tendency for in vivo effects of propionate to alter hepatic gene expression in mid-lactation cows and neonatal calves, which are consistent with a feed-forward effect of propionate to regulate its own metabolism toward gluconeogenesis through changes in hepatic PCK1 mRNA.

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.

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 0.5m M for PRPT; 0.25, 0.5, and 1.0m M for CALV; and 0.5 and 1.0m M for IFL. Cells exposed to PRPT had greater PEPCK-C and tended to have greater G6Pase mRNA than control cells. Exposure of cells to 0.25m M PRPT increased expression of PEPCK-C compared with cells exposed to 0.5m M 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.

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.

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.

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.

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.

The aim of the current study was to determine the effects and the mechanisms of inclusion of dietary whey protein, high calcium, and high vitamin D intake with either a high-sucrose or high-fat base diets on body composition of rodents. Male Wistar rats were assigned to either no whey protein, suboptimal calcium (0.25%), and vitamin D (400 IU/kg) diet (LD), or a diet containing whey protein, high calcium (1.5%), and vitamin D (10 000 IU/kg) diet (HD), and either high-fat (40% of energy) or high-sucrose (60%) base diets for 13 weeks. Liver tissue homogenates were used to determine [(14)C]glucose and [(14)C]palmitate oxidation. mRNA expression of enzymes related to energy metabolism in liver, adipose, and muscle, as well as regulators of muscle mass and insulin receptor was assessed. The results demonstrated that there was reduced accumulation of body fat mass (P = .01) and greater lean mass (P = .03) for the HD- compared to LD-fed group regardless of the background diet. There were no consistent differences between the LD and HD groups across background diets in substrate oxidation and mRNA expression for enzymes measured that regulate energy metabolism, myostatin, or muscle vascular endothelial growth factor. However, there was an increase in insulin receptor mRNA expression in muscle in the HD compared to the LD groups. In conclusion, elevated whey protein, calcium, and vitamin D intake resulted in reduced accumulation of body fat mass and increased lean mass, with a commensurate increase in insulin receptor expression, regardless of the level of calories from fat or sucrose.

Hepatocytes monolayers from neonatal calves were used to determine the responses of the cytosolic phosphoenolpyruvate carboxykinase (PCK1) mRNA expression to propionate and direct hormonal cues including cyclic AMP (cAMP), dexamethasone, and insulin. The responses of other key gluconeogenic genes, including mitochondrial phosphoenolpyruvate carboxykinase (PCK2), pyruvate carboxylase (PC), and glucose-6-phosphotase (G6PC), were also measured. Expression of PCK1 was linearly induced with increasing propionate concentrations in media and 2.5 mM propionate increased PCK1 mRNA at 3 and 6h of incubation; however, the induction disappeared at 12 and 24 h. The induction of PCK1 mRNA by propionate was mimicked by 1 mM cAMP, or in combination with 5 µM dexamethasone, but not by dexamethasone alone. The induction of PCK1 mRNA by propionate or cAMP was eliminated by addition of 100 nM insulin. Additionally, expression of PCK2 and PC mRNA was also induced by propionate in a concentration-dependent manner. Consistent with PCK1, propionate-stimulated PCK2 and PC mRNA expression was inhibited by insulin. Expression of G6PC mRNA was neither affected by propionate nor cAMP, dexamethasone, insulin, or their combinations. These findings demonstrate that propionate can directly regulate its own metabolism in bovine calf hepatocytes through upregulation of PCK1, PCK2, and PC mRNA expression.

Specialized trafficking systems in eukaryotic cells serve a critical role in partitioning intracellular proteins between the nucleus and cytoplasm. Cytoplasmic proteins (including chromatin remodeling enzymes and transcription factors) must gain access to the nucleus to exert their functions to properly program fundamental cellular events ranging from cell cycle progression to gene transcription. Knowing that nuclear import mediated by members of the karyopherin α family of transport receptors plays a critical role in regulating development and differentiation, we wanted to determine the identity of proteins that are trafficked by this karyopherin α pathway. To this end, we performed a GST pull-down assay using porcine orthologs of karyopherin α1 (KPNA1) and karyopherin α7 (KPNA7) and prey protein derived from porcine fibroblast cells and used a liquid chromatography and tandem mass spectrometry (LC-MS/MS) approach to determine the identity of KPNA1 and KPNA7 interacting proteins. Our screen revealed that the proteins that interact with KPNA1 and KPNA7 are generally nuclear proteins that possess nuclear localization signals. We further validated two candidate proteins from this screen and showed that they are able to be imported into the nucleus in vivo and also interact with members of the karyopherin α family of proteins in vitro. Our results also reveal the utility of using a GST pull-down approach coupled with LC-MS/MS to screen for protein interaction partners in a non-traditional model system.

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.

Dried corn distillers grains with sol- ubles (DDGS) fed to swine may adversely affect car- cass quality due to the high concentration of unsatu- rated 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) with DDGS feeding and were 65.07, 69.75, and 74.25 for 0, 20, and 40% DDGS, respectively. Addition of CLA de- creased (P ≤ 0.05) outer layer backfat iodine values from 71.11 to 68.31. Diets containing DDGS decreased (P ≤ 0.05) percent lean tissue contained in bacon from 48% for controls to 38% for pigs fed 40%. Abundance of fatty acid synthase, carnitine palmitoyl transferase Ia, acetyl-CoA-carboxylase, stearoyl-CoA desaturase, and glycerol-3-phosphate dehydrogenase mRNA in adi- pose or liver were not different (P > 0.05) for pigs fed DDGS. Feeding CLA decreased (P ≤ 0.05) the ∆ 9 de- saturase index in adipose tissue. The data indicate that decreased carcass firmness with DDGS feeding is not reflected by changes in lipogenic gene expression. Feed- ing 20% or more DDGS to finishing swine decreases bacon leanness, but inclusion of 0.6% CLA in the fin- ishing diet can partially reverse these effects.

Coordinated partitioning of intracellular cargoes between nuclear and cytoplasmic compartments is critical for cell survival and differentiation. The karyopherin α/β heterodimer functions to import cytoplasmic proteins that possess classical nuclear localisation signals into the nucleus. Seven karyopherin α subtypes have been identified in mammals. The aim of this study was to determine the relative abundance of transcripts encoding seven karyopherin α subtypes in porcine oocytes and embryos at discrete stages of cleavage development, and to determine the developmental requirements of karypopherin α 7 (KPNA7), an oocyte and cleavage stage embryo-specific karyopherin α subtype. We hypothesised that knockdown of KPNA7 would negatively affect porcine cleavage development. To test this hypothesis, in vitro matured and fertilised porcine oocytes were injected with a double-stranded interfering RNA molecule that targeted KPNA7; nuclei were counted in all embryos 6 days after fertilisation. Embryos injected with KPNA7-interfering RNAs possessed significantly lower cell numbers than their respective control groups (P