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The effect of ractopamine hydrochloride on gene expression in adipose tissues of finishing pigs

The effect of ractopamine hydrochloride on gene expression in adipose tissues of finishing pigs ABSTRACT The long-term effect of feeding the catecholamine analog ractopamine (RAC; ractopamine hydrochloride, Elanco Animal Health, Indianapolis, IN) on the expression of genes involved in energy and lipid metabolism in subcutaneous adipose tissue was studied. Large White pigs (84 kg) were fed corn- and soybean meal-based diets supplemented with 0, 20, or 60 mg/kg of RAC for 14, 28, or 42 d. Expression (mRNA abundance) in adipose tissue of sterol regulatory binding protein-1 (SREBP-1), PPARα, PPARγ2, fatty acid synthase (FAS), glucose transporter 4 (GLUT4), and stearoyl-CoA desaturase was determined by Northern blotting. Feed intakes did not differ, and RAC (20 and 60 mg/kg) improved BW gain at d 14, 28, and 42 (P < 0.05) and increased loin eye area (measured on d 42 only; P < 0.05). Expression of SREBP-1 and PPARγ2 declined (P < 0.05) with RAC by d 28 and 42, whereas expression of PPARα was increased (P < 0.05) on d 14, 28, and 42. After 14 d, expression of FAS and GLUT4 was decreased (P < 0.05) with 60 mg/kg of RAC, whereas both RAC concentrations attenuated FAS expression on d 28 and 42. Overall, adipose tissue stearoyl-CoA desaturase expression was not affected by RAC but showed somewhat less expression (P < 0.15) on d 28 at 60 mg/kg of RAC. Although prolonged, chronic RAC feeding most likely downregulates adipose tissue membrane β-adrenergic receptors, mRNA abundances of anabolic lipid metabolism transcription factors, glucose transporters, and enzymes (SREBP-1, PPARγ2, FAS, GLUT4) were still attenuated up to d 42. Conversely, a transcription factor related to oxidative metabolism expression (PPARα) was enhanced. We conclude that even after 42 d, RAC still decreased expression of lipogenic genes in adipose tissue by yet undefined cyclic adenosine monophosphate-directed mechanisms, but in contemporary lean pigs, this effect is likely of limited practical significance. INTRODUCTION Sterol regulatory element binding protein-1 (SREBP-1) and PPARγ are primary regulatory transcription factors (TF) for de novo fatty acid synthesis or lipogenesis (DNL) and adipogenesis, respectively, in humans, rodents, and pigs (Ding et al., 2000; Gondret et al., 2001; Horton et al., 2002; Otto and Lane, 2005; Hausman et al., 2009); however, in pigs, white adipose tissue (WAT), rather than the liver, is the primary site of DNL (Bergen and Mersmann, 2005). Pigs fed β-adrenergic agonists (BAA), including ractopamine hydrochloride (RAC), exhibited increased rates of lipolysis and a diminution in lipogenic enzyme activity in adipose tissue (Merkel et al., 1987; Peterla and Scanes, 1990). This lipolytic effect was attributed to activation of membrane β-adrenergic receptors (BAR), synthesis of cyclic adenosine monophosphate (cAMP), and consequent modulation of lipolytic enzymes in animals and cell lines (Peterla and Scanes, 1990; Dickerson-Weber et al., 1992; Mersmann, 1998; Bergen, 2001; Lefkowitz, 2007;,Hausman et al., 2009). Activities of lipogenic enzymes were shown to be regulated via transcriptional control (Sul and Wang, 1998; Griffin and Sul, 2004), and hepatic fatty acid synthase (FAS) gene expression can be attenuated by cAMP (Oskouian et al., 1996; Rangan et al., 1996; Yellaturu et al., 2005). Any long-term metabolic response to RAC, however, would be dependent on the extent of downregulation of adipose BAR during chronic feeding of RAC (Dunshea et al., 1993, 1998; Liu et al., 1994; Liang and Mills, 2002). Pigs fed RAC have not consistently shown decreased final carcass lipid content (Dunshea et al., 1998). The lack of a consistent RAC effect of final carcass lipid content was attributed to BAR downregulation by the chronic administration of a BAA (Liang and Mills, 2002; Sillence, 2004). The work reported herein was initiated to determine mRNA abundance of the TF, transporters, and enzymes involved in lipid synthesis and oxidation in porcine WAT at timed intervals (14, 28, and 42 d) during a finishing trial with pigs fed 0, 20, or 60 mg/kg of RAC. This experimental approach also provided an opportunity to investigate the physiological effects of chronic RAC feeding and putative BAR downregulation on lipogenic gene expression during chronic RAC feeding in porcine WAT (Dunshea et al., 1993, 1998; Liu et al., 1994; Liang and Mills, 2002). MATERIALS AND METHODS The animal study was conducted at Elanco Animal Health Laboratories (Eli Lilly and Co.) in Greenfield, IN; pig care and feeding, humane slaughter, and tissue sampling protocols were approved by the Lilly Research Laboratories Institutional Animal Care and Use Committee. Animals and Experimental Feeding Study Thirty-five finishing pigs (Yorkshire × Large White Landrace maternal line, crossbred castrated males weighing approximately 84 kg; Pig Improvement Co., Cardinal Farms, Waynetown, IN) were fed an identical 16% CP (N × 6.25) ground corn-soybean meal diet supplemented with all the necessary vitamins and minerals (NRC, 1999) and containing either 0, 20, or 60 mg/kg of 1-(4 hydroxyphenyl)-2-[1 methyl-3(4 hydroxyphenyl) propylamino] (RAC-HCl, Elanco Animal Health, Greenfield, IN) in ethanol for up to 42 d. Pigs were individually housed and fed in 1.8 × 3.0 m pens. During the experimental period, pigs were fed one-half of their daily feed allotment at 0730 and 1530 h. Pigs were weighed at the initiation of the trial and just before each time of slaughter. Pigs were not fasted before slaughter but were fed at 0730 h and then slaughtered beginning 1 h after feeding. Pigs were killed by stunning followed by exsanguination on d 14 (11 pigs), 28 (12 pigs), and 42 (12 pigs). Approximately 30 g of subcutaneous WAT (middle layer) from the loin area was immediately harvested and stored (Reiter et al., 2007). In addition, carcass backfat widths (d 28 and 42) and loin eye areas (d 42 only) were measured. This experiment was not intended to be a production trial, but these carcass measurements were obtained to complement the gene expression results. RNA Isolation from Adipose Tissues Total RNA was isolated using TriZol reagent (Invitrogen, La Jolla, CA, or Sigma, St. Louis, MO), and RNA integrity was assessed electrophoretically (denaturing conditions) as described previously for adipose tissue in our laboratory (Reiter et al., 2007). The RNA samples that showed a prominent 28S, lesser 18S, and minimal small RNA peaks were deemed acceptable, whereas degraded RNA samples were not used. Acceptable RNA samples were then measured spectrophotometrically at 260 nm and stored at −80°C in RNA-Secure (Ambion Inc., Austin, TX). The RNA was then used for gene expression analyses of FAS, SREBP-1, GLUT4, SCD, PPARα, PPARγ2, and β-actin by using Northern blotting. Gene Expression Assays Using Northern Blotting Porcine SREBP-1/ADD1 (adipocyte determination and differentiation factor 1) cDNA (AF102873) in pAMP-1 was provided by H. J. Mersmann, (USDA/Baylor Medical Center, Houston, TX). This SREBP-1 cDNA probe cannot distinguish between the SREBP-1a and SREBP-1c isoforms, but in animal tissues, SREBP-1c mRNA abundance is essentially 10-fold that of SREBP-1a and determinations of SREBP-1 mRNA will principally reflect changes in SREBP-1c expression (Shimomura et al., 1997; Horton, 2002). Complementary DNA fragments were derived for FAS, GLUT4, SCD, PPARγ2, and β-actin with reverse-transcription (RT) PCR using porcine RNA, followed by amplification with a microbial system (Reiter et al., 2007). All RT-PCR reactions were conducted with a OneStep RT-PCR kit (Qiagen Inc., Valencia, CA) and a PTC −100 programmable thermal cycler (MJ Research Inc., Waltham, MA), with the primer sequences and amplicon sizes provided in Table 1. Products from the RT-PCR reactions were gel-purified and applied to Wizard Plus SV Miniprep columns (Promega Corporation, Madison, WI) for final purification. Purified amplicons were ligated into pCR II-TOPO cloning vectors (Invitrogen), and all ligated vectors were used to transform TOP 10F′ Escherichia coli cells (TOPO-TA cloning kit, Invitrogen). Plasmid DNA containing the various inserts was prepared with Maxi kits (Qiagen Inc.) and subsequently sequenced. Sequences for cDNA probes or inserts were reconfirmed just before Northern blotting. Sequencing was accomplished with either automated capillary sequencers (Beckman Coulter Inc., Fullerton, CA, or Applied Biosystems, Foster City, CA) or the cycleSEQ-farOUT polymerase kit (Display Systems Biotech, Copenhagen, Denmark). All probes were checked for a linear response with respect to total RNA by using preliminary Northern blotting, as described previously (Reiter et al., 2007). For cDNA probe preparation, inserts for FAS, GLUT4, SCD, PPARγ2, and β-actin were first excised from their pCR-II vectors with EcoRI; Not1 and Sal1 were used to excise the SREBP-1 insert from pAMP-1. Restriction digests were run on 1.2% low-melting agarose gels, and isolated SREBP-1, FAS, GLUT4, SCD, PPARγ2, and β-actin inserts were purified with a Wizard DNA purification kit (Promega Corporation). From the purified inserts, [32P]cDNA probes were generated with [α-32P]deoxycytidine triphosphate (ICN, Costa Mesa, CA) using the Prime-a gene labeling system (random priming-Klenow fragment, Promega Corporation) and a column purified before use (ProbeQuant G-50 Micro Column, GE Healthcare, Piscataway, NJ). For PPARα, we used a radiolabeled 60-nucleotide antisense oligomer (Table 1). The PPARα antisense oligomer was 5′-end labeled using T4 polynucleotide kinase (Roche, Indianapolis, IN) and [γ32P]ATP (ICN) and then purified with a ProbeQuant G-25 micro column (GE Healthcare; Reiter et al., 2007). Adipose tissue mRNA abundance was determined with Northern blots (Trayhurn, 1996; Reiter et al., 2007). For the gene expression studies, after immobilizing electrophoresed RNA on Nytran membranes (Schleicher and Schuell, Keene, NH), blots were prehybridized in ULTRAhyb solution (Ambion Inc.), followed by addition of radiolabeled cDNA probes [6 × 106 cpm/mL of ULTRAhyb solution (Ambion Inc.)]. The membranes were hybridized overnight with agitation at 42°C and washed twice with 2× SSC and 0.1% SDS, followed by 2 washes in 0.1× SSC and 0.1% SDS at 42°C. Membranes were first exposed to a Molecular Dynamics storage phosphor screen and then scanned with a Typhoon 9410 Variable Mode imager (GE Healthcare). The resulting images were analyzed using Image Quant version 2.1 software (GE Healthcare; Reiter et al., 2007). Table 1. Primer sequences used for PCR amplification of porcine gene fragments Gene1  Primer  Amplicon size, bp  Transcript size, kb  GenBank accession number  FAS  Forward: 5′-GGATCCGCTGCCAGAGTCGGA-3′  214  10.89  AF296508  Reverse: 5′-AAGCTTCAGCTGAGGGTCCATCGTGTGTCCT-3′  SREBP-1  Forward: 5′-TGTGACCTCGCAGATCCAGC-3′  515  4.2  AF102873     Reverse: 5′-GCGAATGTAGTCGATGGCCT-3′  162  4.8  NM_005603  SCD  Forward: 5′-GTGCTTAATGCCACCTGGCTC-3′  162  4.9  NM_005603  Reverse: 5′-TGGCAGAGTAGTCATAGGGAAAGGA-3′  PPARγ  Forward: 5′-CTGTTCCATGCTGTTATGGG-3′  1,539  1.9  AF103946  Reverse: 5′-CTGCTCTAGCTAGTACAAGTCC-3′  GLUT4  Forward: 5′-CCAACAGATAGGCTCCGAAG-3′  520  3.39  AF141956  Reverse: 5′-TGGCCAGTTGGTTGAGCGTC-3′  PPARα  Antisense oligomer: 5′-GGCATCTTCTAGGTCATGTTCACATGTGAGGATTTCTGCTTTCAGTTTGGCTTTTTCAGA-3′  60 nucleotide  8.9  AF228696  β-Actin  Forward: 5′-CATCCTGACCCTCAAGTACCC-3′  245  2.1  AF054837  Reverse: 5′-CCGATGTCGAAGTGGTGGTG-3′  Gene1  Primer  Amplicon size, bp  Transcript size, kb  GenBank accession number  FAS  Forward: 5′-GGATCCGCTGCCAGAGTCGGA-3′  214  10.89  AF296508  Reverse: 5′-AAGCTTCAGCTGAGGGTCCATCGTGTGTCCT-3′  SREBP-1  Forward: 5′-TGTGACCTCGCAGATCCAGC-3′  515  4.2  AF102873     Reverse: 5′-GCGAATGTAGTCGATGGCCT-3′  162  4.8  NM_005603  SCD  Forward: 5′-GTGCTTAATGCCACCTGGCTC-3′  162  4.9  NM_005603  Reverse: 5′-TGGCAGAGTAGTCATAGGGAAAGGA-3′  PPARγ  Forward: 5′-CTGTTCCATGCTGTTATGGG-3′  1,539  1.9  AF103946  Reverse: 5′-CTGCTCTAGCTAGTACAAGTCC-3′  GLUT4  Forward: 5′-CCAACAGATAGGCTCCGAAG-3′  520  3.39  AF141956  Reverse: 5′-TGGCCAGTTGGTTGAGCGTC-3′  PPARα  Antisense oligomer: 5′-GGCATCTTCTAGGTCATGTTCACATGTGAGGATTTCTGCTTTCAGTTTGGCTTTTTCAGA-3′  60 nucleotide  8.9  AF228696  β-Actin  Forward: 5′-CATCCTGACCCTCAAGTACCC-3′  245  2.1  AF054837  Reverse: 5′-CCGATGTCGAAGTGGTGGTG-3′  1FAS = fatty acid synthase; SREBP-1 = sterol regulatory binding protein-1; SCD = stearoyl-CoA desaturase; GLUT4 = glucose transporter 4. View Large Table 1. Primer sequences used for PCR amplification of porcine gene fragments Gene1  Primer  Amplicon size, bp  Transcript size, kb  GenBank accession number  FAS  Forward: 5′-GGATCCGCTGCCAGAGTCGGA-3′  214  10.89  AF296508  Reverse: 5′-AAGCTTCAGCTGAGGGTCCATCGTGTGTCCT-3′  SREBP-1  Forward: 5′-TGTGACCTCGCAGATCCAGC-3′  515  4.2  AF102873     Reverse: 5′-GCGAATGTAGTCGATGGCCT-3′  162  4.8  NM_005603  SCD  Forward: 5′-GTGCTTAATGCCACCTGGCTC-3′  162  4.9  NM_005603  Reverse: 5′-TGGCAGAGTAGTCATAGGGAAAGGA-3′  PPARγ  Forward: 5′-CTGTTCCATGCTGTTATGGG-3′  1,539  1.9  AF103946  Reverse: 5′-CTGCTCTAGCTAGTACAAGTCC-3′  GLUT4  Forward: 5′-CCAACAGATAGGCTCCGAAG-3′  520  3.39  AF141956  Reverse: 5′-TGGCCAGTTGGTTGAGCGTC-3′  PPARα  Antisense oligomer: 5′-GGCATCTTCTAGGTCATGTTCACATGTGAGGATTTCTGCTTTCAGTTTGGCTTTTTCAGA-3′  60 nucleotide  8.9  AF228696  β-Actin  Forward: 5′-CATCCTGACCCTCAAGTACCC-3′  245  2.1  AF054837  Reverse: 5′-CCGATGTCGAAGTGGTGGTG-3′  Gene1  Primer  Amplicon size, bp  Transcript size, kb  GenBank accession number  FAS  Forward: 5′-GGATCCGCTGCCAGAGTCGGA-3′  214  10.89  AF296508  Reverse: 5′-AAGCTTCAGCTGAGGGTCCATCGTGTGTCCT-3′  SREBP-1  Forward: 5′-TGTGACCTCGCAGATCCAGC-3′  515  4.2  AF102873     Reverse: 5′-GCGAATGTAGTCGATGGCCT-3′  162  4.8  NM_005603  SCD  Forward: 5′-GTGCTTAATGCCACCTGGCTC-3′  162  4.9  NM_005603  Reverse: 5′-TGGCAGAGTAGTCATAGGGAAAGGA-3′  PPARγ  Forward: 5′-CTGTTCCATGCTGTTATGGG-3′  1,539  1.9  AF103946  Reverse: 5′-CTGCTCTAGCTAGTACAAGTCC-3′  GLUT4  Forward: 5′-CCAACAGATAGGCTCCGAAG-3′  520  3.39  AF141956  Reverse: 5′-TGGCCAGTTGGTTGAGCGTC-3′  PPARα  Antisense oligomer: 5′-GGCATCTTCTAGGTCATGTTCACATGTGAGGATTTCTGCTTTCAGTTTGGCTTTTTCAGA-3′  60 nucleotide  8.9  AF228696  β-Actin  Forward: 5′-CATCCTGACCCTCAAGTACCC-3′  245  2.1  AF054837  Reverse: 5′-CCGATGTCGAAGTGGTGGTG-3′  1FAS = fatty acid synthase; SREBP-1 = sterol regulatory binding protein-1; SCD = stearoyl-CoA desaturase; GLUT4 = glucose transporter 4. View Large Statistical Analyses Northern blots were analyzed using SAS software ANOVA procedures (SAS Inst. Inc., Cary, NC). Fixed effects were treatment, time of sampling, and treatment × time interactions. Values different at P < 0.05 were considered significant, and means separation, when necessary, was achieved using the Tukey-Kramer multiple-comparison procedure. Preliminary statistical analysis revealed no treatment effects on β-actin mRNA abundance, and Northern blot results for SREBP-1, FAS, SCD, GLUT4, PPARα, and PPARγ2 were normalized to the determined internal control or reference gene (β-actin) expression, with resulting values referred to as relative gene expression. RESULTS Animal Feeding Trial: Effect of Dietary RAC and Length of Feeding Period on Animal Performance and Carcass Characteristics All pigs were fed individually, and individual feed intake and carcass data were obtained for pigs in this study to relate to gene expression results. For 14 d (group 1), 28 d (group 2), and 42 d (group 3), pigs were fed diets with 0, 20, and 60 mg/kg of RAC, respectively. Overall, daily feed intakes by all pigs throughout the experiment were near 2.0 kg. Performance and carcass data are presented in Table 2; no group × treatment interaction was observed. For all the groups, RAC significantly increased daily BW gain, whereas calculated feed:gain ratios declined with increasing RAC. Animal BW gain did not differ, however, between the 20 and 60 mg/kg of RAC treatments for groups 2 and 3. For all groups, RAC had no effect on backfat thickness. Loin eye areas, measured for group 3 only, were increased (P < 0.02) by RAC, but they did not differ between 20 and 60 mg/kg of RAC. Table 2. Performance and carcass data for pigs fed 0, 20, and 60 mg/kg of ractopamine1 Item  Control, 0 mg/kg  20 mg/kg  60 mg/kg  Pigs, n  Group  Treatment  Group × treatment  14 d   ADG, g  732.1 ± 23.0a,A  826.4 ± 23.0b,B  900 ± 24.0c,B  35  NS3  0.001  NS   Daily feed intake, kg  1.93  1.93  1.93               Calculated feed:gain2  2.60  2.34  2.14              28 d   ADG, g  630.67 ± 22.0A  738.61 ± 22.0B  757.95 ± 20.0B  24  NS  0.001  NS   Daily feed intake, kg  2.00  2.00  2.00               Calculated feed:gain  3.20  2.70  2.63               Backfat, mm  5.66 ± 0.47a  4.69 ± 0.46ab  4.18 ± 0.46b     NS  0.07  NS  42 d   ADG, g  603.27 ± 23.1a  691.34 ± 23.1b  693.48 ± 23.1b  12     0.05  NS   Daily feed intake, kg  1.98  1.98  1.98               Calculated feed:gain  3.28  2.86  2.86               Backfat, mm  5.94 ± 0.68a  4.96 ± 0.68a  3.92 ± 0.68a        NS  NS   Loin-eye area, cm2  51.35 ± 2.41a  62.49 ± 2.39b  61.35 ± 2.40b        0.02  NS  Item  Control, 0 mg/kg  20 mg/kg  60 mg/kg  Pigs, n  Group  Treatment  Group × treatment  14 d   ADG, g  732.1 ± 23.0a,A  826.4 ± 23.0b,B  900 ± 24.0c,B  35  NS3  0.001  NS   Daily feed intake, kg  1.93  1.93  1.93               Calculated feed:gain2  2.60  2.34  2.14              28 d   ADG, g  630.67 ± 22.0A  738.61 ± 22.0B  757.95 ± 20.0B  24  NS  0.001  NS   Daily feed intake, kg  2.00  2.00  2.00               Calculated feed:gain  3.20  2.70  2.63               Backfat, mm  5.66 ± 0.47a  4.69 ± 0.46ab  4.18 ± 0.46b     NS  0.07  NS  42 d   ADG, g  603.27 ± 23.1a  691.34 ± 23.1b  693.48 ± 23.1b  12     0.05  NS   Daily feed intake, kg  1.98  1.98  1.98               Calculated feed:gain  3.28  2.86  2.86               Backfat, mm  5.94 ± 0.68a  4.96 ± 0.68a  3.92 ± 0.68a        NS  NS   Loin-eye area, cm2  51.35 ± 2.41a  62.49 ± 2.39b  61.35 ± 2.40b        0.02  NS  a–cWithin a row, means not sharing common superscript are different at P < 0.05. A,BWithin a row, means not sharing common superscript are different at P < 0.01. 1Yorkshire × Large White Landrace maternal line. 2Calculated feed efficiencies (feed:gain) parallel the ADG because fed intakes were identical per day for each pig in the 14-, 28-, and 42-d groups. 3NS = not significant. View Large Table 2. Performance and carcass data for pigs fed 0, 20, and 60 mg/kg of ractopamine1 Item  Control, 0 mg/kg  20 mg/kg  60 mg/kg  Pigs, n  Group  Treatment  Group × treatment  14 d   ADG, g  732.1 ± 23.0a,A  826.4 ± 23.0b,B  900 ± 24.0c,B  35  NS3  0.001  NS   Daily feed intake, kg  1.93  1.93  1.93               Calculated feed:gain2  2.60  2.34  2.14              28 d   ADG, g  630.67 ± 22.0A  738.61 ± 22.0B  757.95 ± 20.0B  24  NS  0.001  NS   Daily feed intake, kg  2.00  2.00  2.00               Calculated feed:gain  3.20  2.70  2.63               Backfat, mm  5.66 ± 0.47a  4.69 ± 0.46ab  4.18 ± 0.46b     NS  0.07  NS  42 d   ADG, g  603.27 ± 23.1a  691.34 ± 23.1b  693.48 ± 23.1b  12     0.05  NS   Daily feed intake, kg  1.98  1.98  1.98               Calculated feed:gain  3.28  2.86  2.86               Backfat, mm  5.94 ± 0.68a  4.96 ± 0.68a  3.92 ± 0.68a        NS  NS   Loin-eye area, cm2  51.35 ± 2.41a  62.49 ± 2.39b  61.35 ± 2.40b        0.02  NS  Item  Control, 0 mg/kg  20 mg/kg  60 mg/kg  Pigs, n  Group  Treatment  Group × treatment  14 d   ADG, g  732.1 ± 23.0a,A  826.4 ± 23.0b,B  900 ± 24.0c,B  35  NS3  0.001  NS   Daily feed intake, kg  1.93  1.93  1.93               Calculated feed:gain2  2.60  2.34  2.14              28 d   ADG, g  630.67 ± 22.0A  738.61 ± 22.0B  757.95 ± 20.0B  24  NS  0.001  NS   Daily feed intake, kg  2.00  2.00  2.00               Calculated feed:gain  3.20  2.70  2.63               Backfat, mm  5.66 ± 0.47a  4.69 ± 0.46ab  4.18 ± 0.46b     NS  0.07  NS  42 d   ADG, g  603.27 ± 23.1a  691.34 ± 23.1b  693.48 ± 23.1b  12     0.05  NS   Daily feed intake, kg  1.98  1.98  1.98               Calculated feed:gain  3.28  2.86  2.86               Backfat, mm  5.94 ± 0.68a  4.96 ± 0.68a  3.92 ± 0.68a        NS  NS   Loin-eye area, cm2  51.35 ± 2.41a  62.49 ± 2.39b  61.35 ± 2.40b        0.02  NS  a–cWithin a row, means not sharing common superscript are different at P < 0.05. A,BWithin a row, means not sharing common superscript are different at P < 0.01. 1Yorkshire × Large White Landrace maternal line. 2Calculated feed efficiencies (feed:gain) parallel the ADG because fed intakes were identical per day for each pig in the 14-, 28-, and 42-d groups. 3NS = not significant. View Large Northern Analysis of mRNA Abundance of the SREBP-1, FAS, GLUT4, SCD, PPARα, and PPARγ2 Genes in Porcine Adipose Tissue Summarized results (statistical comparisons of the 0, 20, and 60 mg/kg of RAC feeding at 14, 28, and 42 d) are presented in Figures 1 and 2, with representative Northern blots provided in the supplemental material (http://jas.fass.org/content/vol89/issue4/). On d 14, feeding RAC reduced mRNA abundances of FAS and GLUT4 (P < 0.05) for the 60 mg/kg of RAC treatment, but not the 20 mg/kg of RAC treatment (Figure 2), whereas the effect of RAC on SREBP-1 on d 14 was not clear because of partial degradation of RNA in the blots. By d 28, mRNA abundances for SREBP-1, FAS, and GLUT4 were decreased for 20 and 60 mg/kg of RAC (Figures 1 and 2). Not only did FAS mRNA abundance decline at d 28 compared with d 14, but FAS mRNA abundances for 20 and 60 mg/kg of RAC were similar. On d 28 and 42 for SREBP-1 and on d 28 for GLUT4, gene expression attenuation was less (P < 0.01) for 20 mg/kg of RAC than for 60 mg/kg of RAC (Figures 1 and 2). On d 42, the expression responses by FAS and SREBP-1 mirrored the expression responses for d 28 (Figures 1 and 2). The blots of GLUT4 mRNA abundance for d 42 were not acceptable for analysis (mRNA degradation) and were not repeated. In the present study, 20 and 60 mg/kg of RAC did not affect mRNA abundances of SCD at d 14, 28, and 42; however, on d 28, at 60 mg/kg of RAC, the attenuation of SCD expression approached significance (P < 0.13; Figure 2). Overall expression (mRNA abundance) of PPARα (a TF that regulates lipid oxidation) was increased (P < 0.01) for both treatments at d 14, 28, and 42. Expression in pigs fed 60 mg/kg of RAC was greater than for pigs fed 20 mg/kg of RAC (P < 0.01, d 14 and 42; P < 0.05, d 28; Figure 1). Expression (mRNA abundance) was not affected on d 14 for PPARγ2, but thereafter, PPARγ2 expression was decreased by RAC for both treatments. The PPARγ2 mRNA abundances were less at 60 mg/kg than at 20 mg/kg of RAC, and both were less than for the controls (Figure 1). Figure 1. View largeDownload slide Effect of feeding 0, 20, and 60 mg/kg of ractopamine hydrochloride to pigs on mRNA abundance in subcutaneous adipose tissue for sterol regulatory binding protein (SREBP-1; panel A; 4.2 kb), PPARα (panel B; 8.9 kb), and PPARγ2 (panel C; 1.9 kb). Tissue RNA was extracted and Northern blotting was conducted as described in the Materials and Methods section. Actual blots are presented in the supplemental material (http://jas.fass.org/content/vol89/issue4/). Expression data (normalized using β-actin as the reference gene; referred to as relative expression) are plotted for each sampling time for all 3 ractopamine concentrations (0, 20, and 60 mg/kg; n = 3). Columns not sharing a common lowercase letter (a–c, within a gene and day category) were significantly different at P < 0.05. Figure 1. View largeDownload slide Effect of feeding 0, 20, and 60 mg/kg of ractopamine hydrochloride to pigs on mRNA abundance in subcutaneous adipose tissue for sterol regulatory binding protein (SREBP-1; panel A; 4.2 kb), PPARα (panel B; 8.9 kb), and PPARγ2 (panel C; 1.9 kb). Tissue RNA was extracted and Northern blotting was conducted as described in the Materials and Methods section. Actual blots are presented in the supplemental material (http://jas.fass.org/content/vol89/issue4/). Expression data (normalized using β-actin as the reference gene; referred to as relative expression) are plotted for each sampling time for all 3 ractopamine concentrations (0, 20, and 60 mg/kg; n = 3). Columns not sharing a common lowercase letter (a–c, within a gene and day category) were significantly different at P < 0.05. Figure 2. View largeDownload slide Effect of feeding 0, 20, and 60 mg/kg of ractopamine hydrochloride to pigs on mRNA abundance in subcutaneous adipose tissue for fatty acid synthase (FAS; panel A; 10.9 kb), stearoyl-CoA desaturase (SCD; panel B; 4.9 kb), and glucose transporter 4 (GLUT4; panel C; 3.4 kb). Tissue RNA was extracted and Northern blotting was conducted as described in the Materials and Methods section. Actual blots are presented in the supplemental material (http://jas.fass.org/content/vol89/issue4/). Expression data (normalized using β-actin as the reference gene; referred to as relative expression) are plotted for each sampling time for all 3 ractopamine concentrations (0, 20, and 60 mg/kg; n = 3). Columns not sharing a common lowercase letter (a–c, within a gene and day category) were significantly different at P < 0.05. Figure 2. View largeDownload slide Effect of feeding 0, 20, and 60 mg/kg of ractopamine hydrochloride to pigs on mRNA abundance in subcutaneous adipose tissue for fatty acid synthase (FAS; panel A; 10.9 kb), stearoyl-CoA desaturase (SCD; panel B; 4.9 kb), and glucose transporter 4 (GLUT4; panel C; 3.4 kb). Tissue RNA was extracted and Northern blotting was conducted as described in the Materials and Methods section. Actual blots are presented in the supplemental material (http://jas.fass.org/content/vol89/issue4/). Expression data (normalized using β-actin as the reference gene; referred to as relative expression) are plotted for each sampling time for all 3 ractopamine concentrations (0, 20, and 60 mg/kg; n = 3). Columns not sharing a common lowercase letter (a–c, within a gene and day category) were significantly different at P < 0.05. DISCUSSION We have conducted a series of experiments on the effect of RAC on expression of lipogenic genes and related TF. The initial work used enzyme activity assays to study the effect of RAC on porcine WAT lipid metabolism (Merkel et al., 1987). The study by Reiter et al. (2007) was designed to assess the role of porcine genotypes (Duroc vs. Pietrains) on lipogenic gene expression and on differential effects of RAC on lipogenic gene expression in skeletal muscle and adipose tissues in these 2 genotypes after a complete finishing period. The present experiment was designed specifically to study lipogenic gene expression patterns in WAT of Large White Landrace pigs at a constant daily energy intake, over a 42-d feeding period, with tissue samplings at 14, 28, and 42 d. Expression of lipogenic enzymes and regulatory TF is sensitive to variations in dietary energy consumption (Sul and Wang, 1998). In this study, any effects of feed intake were negated by providing pigs the same amount of feed, irrespective of RAC treatment. All feed was consumed by the pigs and weigh-backs were not required. Hence, the observed effects of RAC on gene expression, performance, and carcass characteristics cannot be attributed to feed intake differences among treatment groups. In the present study, with pigs consuming identical amounts of DM for the 0, 20, and 60 mg/kg of RAC diets, respectively, daily BW gains were enhanced in the 20 and 60 mg/kg of RAC experimental groups and calculated feed efficiency was improved by feeding RAC. The expected positive effect of RAC on lean deposition was evident (Bergen et al., 1989; Mersmann, 1998; Armstrong et al., 2004; Mimbs et al., 2005) from the increased loin eye area after 42 d. The pigs used here were lean, exhibiting only 4 to 6 mm of measurable backfat thickness at slaughter, and backfat measures were unaffected by RAC. In this study, RAC changed mRNA abundance of both regulatory genes and genes of enzymes involved in fatty acid synthesis in porcine WAT, but these changes appeared to be dependent on both dietary RAC content and length of treatment. Here, mRNA abundance of SREBP-1 was apparently not affected by RAC on d 14 (the data were marginal because of RNA degradation in the blots and are not given) but was decreased at d 28 and 42. Likewise, the expression of PPARγ2, an important regulatory gene for adipogenesis and enhanced lipid storage (Otto and Lane, 2005), was not affected by RAC treatment after 2 wk. Thereafter, for d 28 and 42, RAC decreased PPARγ2 expression for both RAC treatments. The 60 mg/kg of RAC treatment (which is approximately 7 times greater than the recommended RAC concentrations in commercial swine feeding) resulted in less PPARγ2 expression beyond that noted for 20 mg/kg of RAC. Overall, the responses of SREBP-1 and PPARγ2 to RAC feeding were remarkably similar in that expressions of these TF were not affected on d 14, but continued chronic RAC feeding resulted in reduced expression of these TF at d 28 and 42. The present 42-d results are in agreement with our previous work in pigs in which we reported less WAT SREBP-1 and FAS expression after 52 d of RAC feeding (Reiter et al., 2007). However, Liu et al. (1994) reported that mRNA abundance of acetyl CoA carboxylase (a lipogenic enzyme) was not markedly changed in WAT by feeding RAC to pigs up to 24 d. In the present study, PPARα expression was increased with RAC treatment. This TF is associated with oxidative capacity of a tissue (Desvergne et al., 2006). The increase in PPARα expression and marked attenuation of PPARγ2 expression and expression of other lipogenic genes may, in part, reflect the “repartitioning” effect of RAC on adipose fat deposition. Fatty acid synthase mRNA abundance was decreased on d 14 (60 mg/kg only), d 28, and d 42 (20 and 60 mg/kg); abundance of GLUT4 mRNA was decreased at d 14 (60 mg/kg only) and d 28 (20 and 60 mg/kg), whereas SCD expression was only slightly affected at d 28. Sterol regulatory binding protein-1c is a major regulatory determinant of tissue differential lipogenic capacity in mammalian and avian species (Gondret et al., 2001; Horton et al., 2002; Shimano, 2002). In this work, we could demonstrate a decreased abundance for SREBP-1 only for d 28 and 42; hence, in pigs fed RAC, decreased expression of FAS on d 14 without parallel changes in SREBP-1c mRNA abundance could not be clearly evaluated. However, for d 28 and 42, RAC decreased the mRNA abundance of both FAS and SREBP-1 in WAT. These results support other work on parallel expression of SREBP-1 and FAS in porcine adipose tissue (Gondret et al., 2001; Reiter et al., 2007) and agree with the work in rodents (Osborne, 2000; Horton et al., 2002). Evidence also exists that mRNA abundance of SREBP-1c does not always coincide with insulin-mediated changes in lipogenic gene expression in adipose tissues or primary adipocytes (Palmer et al., 2002; Bertile and Raclot, 2004). Recently, the direct effect of SREBP-1c on adipocyte FAS promoter function was assessed, and the results showed that expression of the lipogenic genes was apparently controlled independently of SREBP-1c in rodent adipocytes (Sekiya et al., 2007). Ractopamine may have a direct effect on the regulation of DNL in pigs. Catecholamines (or BAA) and glucagon stimulate the synthesis of cAMP (Sul and Wang, 1998; Lefkowitz, 2006). When hepatocytes are incubated with glucagon or dibutyryl-cAMP, expression of SREBP-1 and its lipogenic target genes is reduced (Foretz et al., 1999; Yellaturu et al., 2005). We showed previously that in adipogenic TA1 cells, RAC decreased mRNA abundance of lipogenic genes in a propranolol (a BAA antagonist)-reversible manner (Dickerson, 1990; Bergen, 2001). Moreover, cAMP appeared directly involved, because nonmetabolizable cAMP analogs also attenuated lipogenic gene expression in TA1 cells in culture (Dickerson, 1990; Bergen, 2001). Expression of SREBP-1c was not measured in those TA1 cell experiments. Other workers have reported that both hepatic FAS and SREBP-1c expression were decreased directly by cAMP (Oskouian et al., 1996; Rangan et al., 1996), and nuclear factor Y has been identified as a possible promoter mediator for cAMP-induced suppression of FAS expression in liver (Roder et al., 2000; Schweizer et al., 2002; Griffin and Sul, 2004; Sul and Smith, 2008). Clarification of the role of SREBP-1c in regulating adipose lipogenic gene expression in pigs fed RAC awaits further studies. From the present observations, the mechanism whereby FAS mRNA abundance is decreased in pigs fed RAC cannot be clearly ascertained. Decreased FAS mRNA abundance may be a consequence of both a direct RAC via cAMP effect on FAS expression and SREBP-1-dependent (after 14 d) regulation of FAS expression in pig adipose tissues. Clearly, these changes in FAS, SREBP-c, and PPARγ2 expression cannot be attributed to differences in feed intake between the 0, 20, and 60 mg/kg of RAC treatments. Feeding RAC decreased GLUT4 (insulin-dependent adipose glucose transporter) mRNA abundance in porcine adipose tissue in concert with cAMP suppression of GLUT4 expression, as shown previously (Kaestner et al., 1991; Cooke and Lane, 1999; Yu et al., 2001; Qi et al., 2009). The mechanism whereby cAMP may modulate GLUT4 expression is not resolved; however, GLUT4 does not possess a cAMP response element consensus sequence in the experimentally determined cAMP response region in the proximal GLUT4 promoter, and the cAMP effect was suggested to be mediated by TF NF1 (Cooke and Lane, 1999). Stearoyl CoA desaturase has long been regarded as a key lipogenic enzyme, particularly in the liver (Flowers and Ntambi, 2008). This enzyme catalyzes the desaturation of palmitic and stearic acids, arising from de novo synthesis or from the diet, to MUFA (Flowers and Ntambi, 2008). In the past, this desaturation activity has been thought to have a primary role in maintaining fluidity of membrane lipid bilayers (Nakamura and Nare, 2004), but more recent studies have also implicated SCD in the development of obesity (Flowers and Ntambi, 2009). In rodents, VLDL synthesis occurs in the liver when using hepatic triacylglycerol. Here again, sizeable desaturation of triacylglycerol fatty acids will result in a more pliable fat that can be transferred to apolipoprotein B, then forming VLDL (Miyazaki and Ntambi, 2008). We had anticipated that in porcine adipose tissue, SCD expression may be attenuated by RAC parallel with FAS and GLUT4 expression, but in the Northern blot study, only a slight attenuation (group 2) was noted for SCD in pigs fed RAC. The Δ9-desaturation of fatty acids in porcine WAT depot lipids may not be as critical in pigs as hepatic Δ9-desaturation of fatty acids in rodents before hepatic VLDL synthesis. The present work thus reveals that at sufficient intakes (20 and 60 mg/kg) of RAC, a decreased expression of specific target genes in lipid metabolism occurs in porcine adipose tissue, particularly the genes involved in DNL and adipogenesis over a 42-d period. Our present study could not corroborate other work (Liu et al., 1994) but agreed with our previous studies (Merkel et al., 1987; Reiter et al., 2007). We observed decreased expression responses in specific target genes despite the likelihood that RAC-BAR binding was downregulated by 40 to 50% in porcine adipose tissue (Liang and Mills, 2002). Recently, Rikard-Bell et al. (2009) used RAC in a pig production trial. Ractopamine was administered in a step-up program to immunocastrated and intact boars and gilts for a 31-d feeding period. Those workers noted positive effects of RAC on final BW and ADG. Furthermore, RAC increased carcass lean content and decreased fat content, particularly in the immunocastrated and intact boars, indicating a positive effect of RAC up to 31 d. Lipogenic genes are extremely sensitive to energy status (Sul and Wang, 1998). Before slaughter, however, food animals are usually placed for several hours in a holding pen where feed is not available. Thus, tissue samples obtained after slaughter may not accurately reflect the patterns of gene expression and enzyme activity [particularly for lipogenic enzymes in porcine adipose tissue; R. A. Merkel (Michigan State University, East Lansing) and W. G. Bergen, unpublished data]. In the present study, pigs were exsanguinated without any intervening period of feed deprivation. It is not clear from the report of Liu et al. (1994) whether pigs underwent any feed deprivation before slaughter; however, their results showed that RAC had no effect on adipose acetyl-CoA carboxylase and malic enzyme activities throughout the experimental period (pretreatment, 1, 8, and 24 d). Furthermore, mRNA abundances of adipose acetyl-CoA carboxylase were not different between any of the sampling time points and treatments (pretreatment, RAC for 1, 8, and 24 d). In retrospect, the data of Liu et al. (1994) likely do not support the conclusion that RAC has, at best, only a short-term effect on lipogenesis (DNL) in porcine adipose tissues. Further work will be necessary to clarify differences between the present work and previous reports. LITERATURE CITED Armstrong T. A. Ivers D. J. Wagner J. R. Anderson D. B. Weldon W. C. Berg E. P. 2004. The effect of dietary ractopamine concentration and duration of feeding on growth performance, carcass characteristics, and meat quality of finishing pigs. J. Anim. Sci.  82: 3245– 3253. [PubMed] Google Scholar CrossRef Search ADS PubMed  Bergen W. G. 2001. The role of cAMP elevating agents and somatotropin on pre- and posttranslational regulation of lipogenesis and lipolysis in Bos taurus and Sus scrofa. Recent Res. Dev. Lipids  5: 47– 59. Bergen W. G. Johnson S. E. Skjaerlund D. M. Babbiker A. S. Ames N. K. Merkel R. A. Anderson D. B. 1989. Muscle protein metabolism in finishing pigs fed ractopamine. J. Anim. Sci.  67: 2255– 2262. [PubMed] Google Scholar CrossRef Search ADS PubMed  Bergen W. G. Mersmann H. J. 2005. Comparative aspects of lipid metabolism: Impact on contemporary research and use of animal models. J. Nutr.  135: 2499– 2502. [PubMed] Google Scholar CrossRef Search ADS PubMed  Bertile F. Raclot T. 2004. Messenger RNA levels of SREBP-1c do not coincide with changes in adipose lipogenic gene expression. Biochem. Biophys. Res. Commun.  325: 827– 834. [PubMed] Google Scholar CrossRef Search ADS PubMed  Cooke D. W. Lane M. D. 1999. Transcription factor NF1 mediates repression of the GLUT4 promoter by cyclic-AMP. Biochem. Biophys. Res. Commun.  260: 600– 604. [PubMed] Google Scholar CrossRef Search ADS PubMed  Desvergne B. Michalik L. Wahli W. 2006. Transcriptional regulation of metabolism. Physiol. Rev.  86: 465– 514. [PubMed] Google Scholar CrossRef Search ADS PubMed  Dickerson, P. S. 1990. The effect of the phenethanolamine ractopamine on lipid metabolism of the adipogenic cell line TA1. PhD Diss.  Michigan State University, East Lansing. Dickerson-Weber P. S. Merkel R. A. Bergen W. G. 1992. Adipogenic cell line TA1: A suitable model to study the effect of β-adrenergic agonists on lipid metabolism. Proc. Exp. Biol. Med.  201: 47– 53. Google Scholar CrossRef Search ADS   Ding S. T. Schinkel A. P. Weber T. E. Mersmann H. J. 2000. Expression of porcine transcription factors and genes related to fatty acid metabolism in different tissues and genetic populations. J. Anim. Sci.  78: 2127– 2134. [PubMed] Google Scholar CrossRef Search ADS PubMed  Dunshea F. R. King R. H. Campbell R. G. Sainz R. D. Kim T. Y. 1993. Interrelationship between sex and ractopamine on protein and lipid deposition in rapidly growing pigs. J. Anim. Sci.  71: 2919– 2930. [PubMed] Google Scholar CrossRef Search ADS PubMed  Dunshea F. R. King R. H. Eason P. J. Campbell R. G. 1998. Interrelationships between dietary ractopamine, energy intake and sex in pigs. Aust. J. Agric. Res.  49: 565– 574. Google Scholar CrossRef Search ADS   Flowers M. T. Ntambi J. M. 2008. Role of stearoyl Co-A desaturase in regulating lipid metabolism. Curr. Opin. Lipidol.  19: 248– 256. [PubMed] Google Scholar CrossRef Search ADS PubMed  Flowers M. T. Ntambi J. M. 2009. Stearoyl-CoA desaturase and its relation to high carbohydrate diets and obesity. Biochim. Biophys. Acta  1791: 85– 91. [PubMed] Google Scholar CrossRef Search ADS PubMed  Foretz M. Pacot C. Dugail I. Lemarchand P. Guichard C. Le Lièpvre X. Berthelier-Lubrano C. Spiegelman B. Kim J. B. Ferré P. Foufelle F. 1999. ADD1/SREBP-1c is required in the activation of hepatic lipogenic gene expression by glucose. Mol. Cell. Biol.  19: 3760– 3768. [PubMed] Google Scholar CrossRef Search ADS PubMed  Gondret F. Ferre P. Dugail I. 2001. ADD-1/SREBP-1 is a major determinant of tissue differential lipogenic capacity in mammalian and avian species. J. Lipid Res.  42: 106– 113. [PubMed] Google Scholar PubMed  Griffin M. J. Sul H. S. 2004. Insulin regulation of fatty acid synthase gene transcription: Roles of USF and SREBP-1c. IUBMB Life  56: 595– 600. [PubMed] Google Scholar CrossRef Search ADS PubMed  Hausman G. J. Dodson M. V. Ajuwon K. Azain M. Barnes K. M. Guan L. L. Jiang Z. Poulos S. P. Sainz R. D. Smith S. Spurlock M. Novakofski J. Fernyhough M. E. Bergen W. G. 2009. Board-invited review: The biology and regulation of preadipocytes and adipocytes in meat animals. J. Anim. Sci.  87: 1218– 1246. [PubMed] Google Scholar CrossRef Search ADS PubMed  Horton J. D. 2002. Sterol regulatory element-binding proteins: Transcriptional regulators of lipid synthesis. Biochem. Soc. Trans.  30: 1091– 1095. [PubMed] Google Scholar CrossRef Search ADS PubMed  Horton J. D. Goldstein J. L. Brown M. S. 2002. SREBPs: Activators of the complete program of cholesterol and fatty acid synthesis in the liver. J. Clin. Invest.  109: 1125– 1131. [PubMed] Google Scholar CrossRef Search ADS PubMed  Kaestner K. H. Flores-Riveros J. R. McLenithan J. C. Janicot M. Lane M. D. 1991. Transcriptional repression of the mouse insulin-responsive glucose transporter (GLUT4) gene by cAMP. Proc. Natl. Acad. Sci. USA  88: 1933– 1937. [PubMed] Google Scholar CrossRef Search ADS   Lefkowitz R. J. 2007. Seven transmembrane receptors: A brief personal retrospective. Biochim. Biophys. Acta  1768: 748– 755. [PubMed] Google Scholar CrossRef Search ADS PubMed  Liang W. Mills S. E. 2002. Quantitative analysis of β-adrenergic receptor subtypes in pig tissues. J. Anim. Sci.  80: 963– 970. [PubMed] Google Scholar CrossRef Search ADS PubMed  Liu C. Y. Grant A. L. Kim K. H. Ji S. Q. Hancock D. L. Anderson D. B. Mills S. E. 1994. Limitations of ractopamine to affect adipose tissue metabolism in swine. J. Anim. Sci.  72: 62– 67. [PubMed] Google Scholar CrossRef Search ADS PubMed  Merkel R. A. Dickerson P. S. Johnson S. E. Burkett R. L. Burnett R. J. Schroeder A. L. Bergen W. G. Anderson D. B. 1987. The effect of ractopamine on lipid metabolism in pigs. Fed. Proc.  46: 1177A. (Abstr.) Mersmann H. J. 1998. Overview of the effects of β-adrenergic receptor agonists on animals including mechanisms of action. J. Anim. Sci.  76: 160– 172. [PubMed] Google Scholar CrossRef Search ADS PubMed  Mimbs K. J. Pringle T. D. Azain M. J. Meers S. A. Armstrong T. A. 2005. Effects of ractopamine on performance and composition of pigs phenotypically sorted into fat and lean groups. J. Anim. Sci.  83: 1361– 1369. [PubMed] Google Scholar CrossRef Search ADS PubMed  Miyazaki, M., and J. M. Ntambi 2008. Fatty acid desaturation and chain elongation in mammals. Pages 191–211 in Biochemistry of Lipids, Lipoproteins and Membranes.  5th ed. D. E. Vance and J. E. Vance ed. Elsevier, Amsterdam, the Netherlands. Google Scholar CrossRef Search ADS   Nakamura M. T. Nare T. Y. 2004. Structure, function, and dietary regulation of Δ6, Δ5 and Δ9 desaturases. Annu. Rev. Nutr.  24: 345– 376. [PubMed] Google Scholar CrossRef Search ADS PubMed  NRC 1999. Nutrient Requirements of Swine.  10th rev. ed. Natl. Acad. Press, Washington DC. Osborne T. F. 2000. Sterol regulatory element-binding proteins (SREBPs): Key regulators of nutritional homeostasis and insulin action. J. Biol. Chem.  275: 32379– 32382. [PubMed] Google Scholar CrossRef Search ADS PubMed  Oskouian B. Rangan V. S. Smith S. 1996. Transcriptional regulation of the rat fatty acid synthase gene: Identification of functional analysis of positive and negative effectors of basal transcription. Biochem. J.  317: 257– 265. [PubMed] Google Scholar CrossRef Search ADS PubMed  Otto T. C. Lane M. D. 2005. Adipose development: From stem cell to adipocyte. Crit. Rev. Biochem. Mol. Biol.  40: 229– 242. [PubMed] Google Scholar CrossRef Search ADS PubMed  Palmer D. G. Rutter G. A. Tavaré J. M. 2002. Insulin-stimulated fatty acid synthase gene expression does not require increased sterol response element binding protein 1 transcription in primary adipocytes. Biochem. Biophys. Res. Commun.  291: 439– 443. [PubMed] Google Scholar CrossRef Search ADS PubMed  Peterla T. A. Scanes C. G. 1990. Effect of β-adrenergic agonists on lipolysis and lipogenesis by porcine adipose tissue in vitro. J. Anim. Sci.  68: 1024– 1029. [PubMed] Google Scholar CrossRef Search ADS PubMed  Qi L. Saberi M. Zmuda E. Wang Y. Altarejos J. Zhang X. Dentin R. Hedrick S. Bandyopadhyay G. Hai T. Olefsky J. Montminy M. 2009. Adipocyte CREB promotes insulin resistance in obesity. Cell Metab.  9: 277– 286. [PubMed] Google Scholar CrossRef Search ADS PubMed  Rangan V. S. Oskouian B. Smith S. 1996. Identification of an inverted CCAAT box motif in the fatty acid synthase gene as an essential element for mediation of transcriptional regulation by cAMP. J. Biol. Chem.  271: 2307– 2312. [PubMed] Google Scholar CrossRef Search ADS PubMed  Reiter S. S. Halsey C. H. C. Stronach B. J. Bartosh J. L. Owsley W. F. Bergen W. G. 2007. Lipid metabolism related gene-expression profiling in liver, skeletal muscle and adipose tissue in crossbred Duroc and Pietrain pigs. Comp. Biochem. Physiol. D Genomics Proteomics  2: 200– 206. [PubMed] Google Scholar CrossRef Search ADS   Rikard-Bell C. Curtis M. A. van Barneveld R. J. Mullan B. P. Edwards A. C. Gannon N. J. Henman D. J. Hughes P. E. Dunshea F. R. 2009. Ractopamine hydrochloride improves growth performance and carcass composition in immunocastrated boars, intact boars, and gilts. J. Anim. Sci.  87: 3536– 3543. [PubMed] Google Scholar CrossRef Search ADS PubMed  Roder K. Wolf S. S. Schweizer M. E. 2000. Dependence of rat spot 14 promoter activity on NF-Y binding to the inverted CCAAT-element at −100. Biosci. Biotechnol. Biochem.  64: 848– 851. [PubMed] Google Scholar CrossRef Search ADS PubMed  Schweizer M. E. Roder K. Zhang L. Wolf S. S. 2002. Transcription factors acting on the promoter of rat fatty acid synthase gene. Biochem. Soc. Trans.  30: 1070– 1072. [PubMed] Google Scholar CrossRef Search ADS PubMed  Sekiya M. Yahagi N. Matsuzaka T. Takeuchi Y. Nakagawa Y. Takahashi H. Okazaki H. Iizuka Y. Ohashi K. Gotoda T. Ishibashi S. Nagai R. Yamazaki T. Kadowaki T. Yamada N. Osuga J. Shimano H. 2007. SREBP-1 independent regulation of lipogenic gene expression in adipocytes. J. Lipid Res.  48: 1581– 1591. [PubMed] Google Scholar CrossRef Search ADS PubMed  Shimano H. 2002. Sterol regulatory element-binding protein family as global regulators of lipid synthetic genes in energy metabolism. Vitam. Horm.  65: 167– 194. [PubMed] Google Scholar CrossRef Search ADS PubMed  Shimomura I. Shimano H. Horton J. D. Goldstein J. L. Brown M. S. 1997. Differential expression of exons 1a and 1c for sterol regulatory element binding protein-1 in human and mouse organs and cultured cells. J. Clin. Invest.  99: 838– 845. [PubMed] Google Scholar CrossRef Search ADS PubMed  Sillence M. N. 2004. Technologies for the control of fat and lean deposition in livestock. Vet. J.  167: 242– 257. [PubMed] Google Scholar CrossRef Search ADS PubMed  Sul, H. S., and S. Smith 2008. Fatty acid synthesis in eukaryotes. Pages 155–190 in Biochemistry of Lipids, Lipoproteins and Membranes.  5th ed. D. E. Vance and J. E. Vance ed. Elsevier, Amsterdam, the Netherlands. Google Scholar CrossRef Search ADS   Sul H. S. Wang D. 1998. Nutritional and hormonal regulation of enzymes in fat synthesis: Studies of fatty acid and mitochondrial glycerol-3-phosphate acyltransferase gene transcription. Annu. Rev. Nutr.  18: 331– 351. [PubMed] Google Scholar CrossRef Search ADS PubMed  Trayhurn P. 1996. Northern blotting. Proc. Nutr. Soc.  55: 583– 589. [PubMed] Google Scholar CrossRef Search ADS PubMed  Yellaturu C. R. Deng X. Cagen L. M. Wilcox H. G. Park E. A. Raghow R. Elam M. B. 2005. Posttranslational processing of SREBP-1 in rat hepatocytes is regulated by insulin and cAMP. Biochem. Biophys. Res. Commun.  332: 174– 180. [PubMed] Google Scholar CrossRef Search ADS PubMed  Yu Z. W. Buren J. Enerback S. Nilsson E. Samuelsson L. Eriksson J. W. 2001. Insulin can enhance GLUT4 gene expression in 3T3-F442A cells and this effect is mimicked by vanadate but counteracted by cAMP and high glucose: Potential implications for insulin resistance. Biochim. Biophys. Acta  1535: 174– 185. [PubMed] Google Scholar CrossRef Search ADS PubMed  Footnotes 1 The work presented herein was supported by the Alabama Agricultural Experiment Station (Auburn), the Michigan Agricultural Experiment Station (Mt. Pleasant), Lilly Research Laboratories (Greenfield, IN), the Upchurch Fund for Excellence in Animal Sciences at Auburn University (Auburn), the Alabama Pork Producers Association (Montgomery), and USDA-National Research Initiative (Washington, DC) grants 93-03336 and 2004-35206-14123 to W. G. Bergen. Special thanks go to David B. Anderson, Emerson L. Potter, and Aubrey L. Schroeder (Lilly Research Laboratories, Elanco Animal Health, Indianapolis, IN) for support during this research. A portion of this work was presented in abstract form at Experimental Biology 2003 (San Diego, CA; April 11–15, 2003). American Society of Animal Science http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Animal Science Oxford University Press

The effect of ractopamine hydrochloride on gene expression in adipose tissues of finishing pigs

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0021-8812
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1525-3163
DOI
10.2527/jas.2010-3269
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21148782
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Abstract

ABSTRACT The long-term effect of feeding the catecholamine analog ractopamine (RAC; ractopamine hydrochloride, Elanco Animal Health, Indianapolis, IN) on the expression of genes involved in energy and lipid metabolism in subcutaneous adipose tissue was studied. Large White pigs (84 kg) were fed corn- and soybean meal-based diets supplemented with 0, 20, or 60 mg/kg of RAC for 14, 28, or 42 d. Expression (mRNA abundance) in adipose tissue of sterol regulatory binding protein-1 (SREBP-1), PPARα, PPARγ2, fatty acid synthase (FAS), glucose transporter 4 (GLUT4), and stearoyl-CoA desaturase was determined by Northern blotting. Feed intakes did not differ, and RAC (20 and 60 mg/kg) improved BW gain at d 14, 28, and 42 (P < 0.05) and increased loin eye area (measured on d 42 only; P < 0.05). Expression of SREBP-1 and PPARγ2 declined (P < 0.05) with RAC by d 28 and 42, whereas expression of PPARα was increased (P < 0.05) on d 14, 28, and 42. After 14 d, expression of FAS and GLUT4 was decreased (P < 0.05) with 60 mg/kg of RAC, whereas both RAC concentrations attenuated FAS expression on d 28 and 42. Overall, adipose tissue stearoyl-CoA desaturase expression was not affected by RAC but showed somewhat less expression (P < 0.15) on d 28 at 60 mg/kg of RAC. Although prolonged, chronic RAC feeding most likely downregulates adipose tissue membrane β-adrenergic receptors, mRNA abundances of anabolic lipid metabolism transcription factors, glucose transporters, and enzymes (SREBP-1, PPARγ2, FAS, GLUT4) were still attenuated up to d 42. Conversely, a transcription factor related to oxidative metabolism expression (PPARα) was enhanced. We conclude that even after 42 d, RAC still decreased expression of lipogenic genes in adipose tissue by yet undefined cyclic adenosine monophosphate-directed mechanisms, but in contemporary lean pigs, this effect is likely of limited practical significance. INTRODUCTION Sterol regulatory element binding protein-1 (SREBP-1) and PPARγ are primary regulatory transcription factors (TF) for de novo fatty acid synthesis or lipogenesis (DNL) and adipogenesis, respectively, in humans, rodents, and pigs (Ding et al., 2000; Gondret et al., 2001; Horton et al., 2002; Otto and Lane, 2005; Hausman et al., 2009); however, in pigs, white adipose tissue (WAT), rather than the liver, is the primary site of DNL (Bergen and Mersmann, 2005). Pigs fed β-adrenergic agonists (BAA), including ractopamine hydrochloride (RAC), exhibited increased rates of lipolysis and a diminution in lipogenic enzyme activity in adipose tissue (Merkel et al., 1987; Peterla and Scanes, 1990). This lipolytic effect was attributed to activation of membrane β-adrenergic receptors (BAR), synthesis of cyclic adenosine monophosphate (cAMP), and consequent modulation of lipolytic enzymes in animals and cell lines (Peterla and Scanes, 1990; Dickerson-Weber et al., 1992; Mersmann, 1998; Bergen, 2001; Lefkowitz, 2007;,Hausman et al., 2009). Activities of lipogenic enzymes were shown to be regulated via transcriptional control (Sul and Wang, 1998; Griffin and Sul, 2004), and hepatic fatty acid synthase (FAS) gene expression can be attenuated by cAMP (Oskouian et al., 1996; Rangan et al., 1996; Yellaturu et al., 2005). Any long-term metabolic response to RAC, however, would be dependent on the extent of downregulation of adipose BAR during chronic feeding of RAC (Dunshea et al., 1993, 1998; Liu et al., 1994; Liang and Mills, 2002). Pigs fed RAC have not consistently shown decreased final carcass lipid content (Dunshea et al., 1998). The lack of a consistent RAC effect of final carcass lipid content was attributed to BAR downregulation by the chronic administration of a BAA (Liang and Mills, 2002; Sillence, 2004). The work reported herein was initiated to determine mRNA abundance of the TF, transporters, and enzymes involved in lipid synthesis and oxidation in porcine WAT at timed intervals (14, 28, and 42 d) during a finishing trial with pigs fed 0, 20, or 60 mg/kg of RAC. This experimental approach also provided an opportunity to investigate the physiological effects of chronic RAC feeding and putative BAR downregulation on lipogenic gene expression during chronic RAC feeding in porcine WAT (Dunshea et al., 1993, 1998; Liu et al., 1994; Liang and Mills, 2002). MATERIALS AND METHODS The animal study was conducted at Elanco Animal Health Laboratories (Eli Lilly and Co.) in Greenfield, IN; pig care and feeding, humane slaughter, and tissue sampling protocols were approved by the Lilly Research Laboratories Institutional Animal Care and Use Committee. Animals and Experimental Feeding Study Thirty-five finishing pigs (Yorkshire × Large White Landrace maternal line, crossbred castrated males weighing approximately 84 kg; Pig Improvement Co., Cardinal Farms, Waynetown, IN) were fed an identical 16% CP (N × 6.25) ground corn-soybean meal diet supplemented with all the necessary vitamins and minerals (NRC, 1999) and containing either 0, 20, or 60 mg/kg of 1-(4 hydroxyphenyl)-2-[1 methyl-3(4 hydroxyphenyl) propylamino] (RAC-HCl, Elanco Animal Health, Greenfield, IN) in ethanol for up to 42 d. Pigs were individually housed and fed in 1.8 × 3.0 m pens. During the experimental period, pigs were fed one-half of their daily feed allotment at 0730 and 1530 h. Pigs were weighed at the initiation of the trial and just before each time of slaughter. Pigs were not fasted before slaughter but were fed at 0730 h and then slaughtered beginning 1 h after feeding. Pigs were killed by stunning followed by exsanguination on d 14 (11 pigs), 28 (12 pigs), and 42 (12 pigs). Approximately 30 g of subcutaneous WAT (middle layer) from the loin area was immediately harvested and stored (Reiter et al., 2007). In addition, carcass backfat widths (d 28 and 42) and loin eye areas (d 42 only) were measured. This experiment was not intended to be a production trial, but these carcass measurements were obtained to complement the gene expression results. RNA Isolation from Adipose Tissues Total RNA was isolated using TriZol reagent (Invitrogen, La Jolla, CA, or Sigma, St. Louis, MO), and RNA integrity was assessed electrophoretically (denaturing conditions) as described previously for adipose tissue in our laboratory (Reiter et al., 2007). The RNA samples that showed a prominent 28S, lesser 18S, and minimal small RNA peaks were deemed acceptable, whereas degraded RNA samples were not used. Acceptable RNA samples were then measured spectrophotometrically at 260 nm and stored at −80°C in RNA-Secure (Ambion Inc., Austin, TX). The RNA was then used for gene expression analyses of FAS, SREBP-1, GLUT4, SCD, PPARα, PPARγ2, and β-actin by using Northern blotting. Gene Expression Assays Using Northern Blotting Porcine SREBP-1/ADD1 (adipocyte determination and differentiation factor 1) cDNA (AF102873) in pAMP-1 was provided by H. J. Mersmann, (USDA/Baylor Medical Center, Houston, TX). This SREBP-1 cDNA probe cannot distinguish between the SREBP-1a and SREBP-1c isoforms, but in animal tissues, SREBP-1c mRNA abundance is essentially 10-fold that of SREBP-1a and determinations of SREBP-1 mRNA will principally reflect changes in SREBP-1c expression (Shimomura et al., 1997; Horton, 2002). Complementary DNA fragments were derived for FAS, GLUT4, SCD, PPARγ2, and β-actin with reverse-transcription (RT) PCR using porcine RNA, followed by amplification with a microbial system (Reiter et al., 2007). All RT-PCR reactions were conducted with a OneStep RT-PCR kit (Qiagen Inc., Valencia, CA) and a PTC −100 programmable thermal cycler (MJ Research Inc., Waltham, MA), with the primer sequences and amplicon sizes provided in Table 1. Products from the RT-PCR reactions were gel-purified and applied to Wizard Plus SV Miniprep columns (Promega Corporation, Madison, WI) for final purification. Purified amplicons were ligated into pCR II-TOPO cloning vectors (Invitrogen), and all ligated vectors were used to transform TOP 10F′ Escherichia coli cells (TOPO-TA cloning kit, Invitrogen). Plasmid DNA containing the various inserts was prepared with Maxi kits (Qiagen Inc.) and subsequently sequenced. Sequences for cDNA probes or inserts were reconfirmed just before Northern blotting. Sequencing was accomplished with either automated capillary sequencers (Beckman Coulter Inc., Fullerton, CA, or Applied Biosystems, Foster City, CA) or the cycleSEQ-farOUT polymerase kit (Display Systems Biotech, Copenhagen, Denmark). All probes were checked for a linear response with respect to total RNA by using preliminary Northern blotting, as described previously (Reiter et al., 2007). For cDNA probe preparation, inserts for FAS, GLUT4, SCD, PPARγ2, and β-actin were first excised from their pCR-II vectors with EcoRI; Not1 and Sal1 were used to excise the SREBP-1 insert from pAMP-1. Restriction digests were run on 1.2% low-melting agarose gels, and isolated SREBP-1, FAS, GLUT4, SCD, PPARγ2, and β-actin inserts were purified with a Wizard DNA purification kit (Promega Corporation). From the purified inserts, [32P]cDNA probes were generated with [α-32P]deoxycytidine triphosphate (ICN, Costa Mesa, CA) using the Prime-a gene labeling system (random priming-Klenow fragment, Promega Corporation) and a column purified before use (ProbeQuant G-50 Micro Column, GE Healthcare, Piscataway, NJ). For PPARα, we used a radiolabeled 60-nucleotide antisense oligomer (Table 1). The PPARα antisense oligomer was 5′-end labeled using T4 polynucleotide kinase (Roche, Indianapolis, IN) and [γ32P]ATP (ICN) and then purified with a ProbeQuant G-25 micro column (GE Healthcare; Reiter et al., 2007). Adipose tissue mRNA abundance was determined with Northern blots (Trayhurn, 1996; Reiter et al., 2007). For the gene expression studies, after immobilizing electrophoresed RNA on Nytran membranes (Schleicher and Schuell, Keene, NH), blots were prehybridized in ULTRAhyb solution (Ambion Inc.), followed by addition of radiolabeled cDNA probes [6 × 106 cpm/mL of ULTRAhyb solution (Ambion Inc.)]. The membranes were hybridized overnight with agitation at 42°C and washed twice with 2× SSC and 0.1% SDS, followed by 2 washes in 0.1× SSC and 0.1% SDS at 42°C. Membranes were first exposed to a Molecular Dynamics storage phosphor screen and then scanned with a Typhoon 9410 Variable Mode imager (GE Healthcare). The resulting images were analyzed using Image Quant version 2.1 software (GE Healthcare; Reiter et al., 2007). Table 1. Primer sequences used for PCR amplification of porcine gene fragments Gene1  Primer  Amplicon size, bp  Transcript size, kb  GenBank accession number  FAS  Forward: 5′-GGATCCGCTGCCAGAGTCGGA-3′  214  10.89  AF296508  Reverse: 5′-AAGCTTCAGCTGAGGGTCCATCGTGTGTCCT-3′  SREBP-1  Forward: 5′-TGTGACCTCGCAGATCCAGC-3′  515  4.2  AF102873     Reverse: 5′-GCGAATGTAGTCGATGGCCT-3′  162  4.8  NM_005603  SCD  Forward: 5′-GTGCTTAATGCCACCTGGCTC-3′  162  4.9  NM_005603  Reverse: 5′-TGGCAGAGTAGTCATAGGGAAAGGA-3′  PPARγ  Forward: 5′-CTGTTCCATGCTGTTATGGG-3′  1,539  1.9  AF103946  Reverse: 5′-CTGCTCTAGCTAGTACAAGTCC-3′  GLUT4  Forward: 5′-CCAACAGATAGGCTCCGAAG-3′  520  3.39  AF141956  Reverse: 5′-TGGCCAGTTGGTTGAGCGTC-3′  PPARα  Antisense oligomer: 5′-GGCATCTTCTAGGTCATGTTCACATGTGAGGATTTCTGCTTTCAGTTTGGCTTTTTCAGA-3′  60 nucleotide  8.9  AF228696  β-Actin  Forward: 5′-CATCCTGACCCTCAAGTACCC-3′  245  2.1  AF054837  Reverse: 5′-CCGATGTCGAAGTGGTGGTG-3′  Gene1  Primer  Amplicon size, bp  Transcript size, kb  GenBank accession number  FAS  Forward: 5′-GGATCCGCTGCCAGAGTCGGA-3′  214  10.89  AF296508  Reverse: 5′-AAGCTTCAGCTGAGGGTCCATCGTGTGTCCT-3′  SREBP-1  Forward: 5′-TGTGACCTCGCAGATCCAGC-3′  515  4.2  AF102873     Reverse: 5′-GCGAATGTAGTCGATGGCCT-3′  162  4.8  NM_005603  SCD  Forward: 5′-GTGCTTAATGCCACCTGGCTC-3′  162  4.9  NM_005603  Reverse: 5′-TGGCAGAGTAGTCATAGGGAAAGGA-3′  PPARγ  Forward: 5′-CTGTTCCATGCTGTTATGGG-3′  1,539  1.9  AF103946  Reverse: 5′-CTGCTCTAGCTAGTACAAGTCC-3′  GLUT4  Forward: 5′-CCAACAGATAGGCTCCGAAG-3′  520  3.39  AF141956  Reverse: 5′-TGGCCAGTTGGTTGAGCGTC-3′  PPARα  Antisense oligomer: 5′-GGCATCTTCTAGGTCATGTTCACATGTGAGGATTTCTGCTTTCAGTTTGGCTTTTTCAGA-3′  60 nucleotide  8.9  AF228696  β-Actin  Forward: 5′-CATCCTGACCCTCAAGTACCC-3′  245  2.1  AF054837  Reverse: 5′-CCGATGTCGAAGTGGTGGTG-3′  1FAS = fatty acid synthase; SREBP-1 = sterol regulatory binding protein-1; SCD = stearoyl-CoA desaturase; GLUT4 = glucose transporter 4. View Large Table 1. Primer sequences used for PCR amplification of porcine gene fragments Gene1  Primer  Amplicon size, bp  Transcript size, kb  GenBank accession number  FAS  Forward: 5′-GGATCCGCTGCCAGAGTCGGA-3′  214  10.89  AF296508  Reverse: 5′-AAGCTTCAGCTGAGGGTCCATCGTGTGTCCT-3′  SREBP-1  Forward: 5′-TGTGACCTCGCAGATCCAGC-3′  515  4.2  AF102873     Reverse: 5′-GCGAATGTAGTCGATGGCCT-3′  162  4.8  NM_005603  SCD  Forward: 5′-GTGCTTAATGCCACCTGGCTC-3′  162  4.9  NM_005603  Reverse: 5′-TGGCAGAGTAGTCATAGGGAAAGGA-3′  PPARγ  Forward: 5′-CTGTTCCATGCTGTTATGGG-3′  1,539  1.9  AF103946  Reverse: 5′-CTGCTCTAGCTAGTACAAGTCC-3′  GLUT4  Forward: 5′-CCAACAGATAGGCTCCGAAG-3′  520  3.39  AF141956  Reverse: 5′-TGGCCAGTTGGTTGAGCGTC-3′  PPARα  Antisense oligomer: 5′-GGCATCTTCTAGGTCATGTTCACATGTGAGGATTTCTGCTTTCAGTTTGGCTTTTTCAGA-3′  60 nucleotide  8.9  AF228696  β-Actin  Forward: 5′-CATCCTGACCCTCAAGTACCC-3′  245  2.1  AF054837  Reverse: 5′-CCGATGTCGAAGTGGTGGTG-3′  Gene1  Primer  Amplicon size, bp  Transcript size, kb  GenBank accession number  FAS  Forward: 5′-GGATCCGCTGCCAGAGTCGGA-3′  214  10.89  AF296508  Reverse: 5′-AAGCTTCAGCTGAGGGTCCATCGTGTGTCCT-3′  SREBP-1  Forward: 5′-TGTGACCTCGCAGATCCAGC-3′  515  4.2  AF102873     Reverse: 5′-GCGAATGTAGTCGATGGCCT-3′  162  4.8  NM_005603  SCD  Forward: 5′-GTGCTTAATGCCACCTGGCTC-3′  162  4.9  NM_005603  Reverse: 5′-TGGCAGAGTAGTCATAGGGAAAGGA-3′  PPARγ  Forward: 5′-CTGTTCCATGCTGTTATGGG-3′  1,539  1.9  AF103946  Reverse: 5′-CTGCTCTAGCTAGTACAAGTCC-3′  GLUT4  Forward: 5′-CCAACAGATAGGCTCCGAAG-3′  520  3.39  AF141956  Reverse: 5′-TGGCCAGTTGGTTGAGCGTC-3′  PPARα  Antisense oligomer: 5′-GGCATCTTCTAGGTCATGTTCACATGTGAGGATTTCTGCTTTCAGTTTGGCTTTTTCAGA-3′  60 nucleotide  8.9  AF228696  β-Actin  Forward: 5′-CATCCTGACCCTCAAGTACCC-3′  245  2.1  AF054837  Reverse: 5′-CCGATGTCGAAGTGGTGGTG-3′  1FAS = fatty acid synthase; SREBP-1 = sterol regulatory binding protein-1; SCD = stearoyl-CoA desaturase; GLUT4 = glucose transporter 4. View Large Statistical Analyses Northern blots were analyzed using SAS software ANOVA procedures (SAS Inst. Inc., Cary, NC). Fixed effects were treatment, time of sampling, and treatment × time interactions. Values different at P < 0.05 were considered significant, and means separation, when necessary, was achieved using the Tukey-Kramer multiple-comparison procedure. Preliminary statistical analysis revealed no treatment effects on β-actin mRNA abundance, and Northern blot results for SREBP-1, FAS, SCD, GLUT4, PPARα, and PPARγ2 were normalized to the determined internal control or reference gene (β-actin) expression, with resulting values referred to as relative gene expression. RESULTS Animal Feeding Trial: Effect of Dietary RAC and Length of Feeding Period on Animal Performance and Carcass Characteristics All pigs were fed individually, and individual feed intake and carcass data were obtained for pigs in this study to relate to gene expression results. For 14 d (group 1), 28 d (group 2), and 42 d (group 3), pigs were fed diets with 0, 20, and 60 mg/kg of RAC, respectively. Overall, daily feed intakes by all pigs throughout the experiment were near 2.0 kg. Performance and carcass data are presented in Table 2; no group × treatment interaction was observed. For all the groups, RAC significantly increased daily BW gain, whereas calculated feed:gain ratios declined with increasing RAC. Animal BW gain did not differ, however, between the 20 and 60 mg/kg of RAC treatments for groups 2 and 3. For all groups, RAC had no effect on backfat thickness. Loin eye areas, measured for group 3 only, were increased (P < 0.02) by RAC, but they did not differ between 20 and 60 mg/kg of RAC. Table 2. Performance and carcass data for pigs fed 0, 20, and 60 mg/kg of ractopamine1 Item  Control, 0 mg/kg  20 mg/kg  60 mg/kg  Pigs, n  Group  Treatment  Group × treatment  14 d   ADG, g  732.1 ± 23.0a,A  826.4 ± 23.0b,B  900 ± 24.0c,B  35  NS3  0.001  NS   Daily feed intake, kg  1.93  1.93  1.93               Calculated feed:gain2  2.60  2.34  2.14              28 d   ADG, g  630.67 ± 22.0A  738.61 ± 22.0B  757.95 ± 20.0B  24  NS  0.001  NS   Daily feed intake, kg  2.00  2.00  2.00               Calculated feed:gain  3.20  2.70  2.63               Backfat, mm  5.66 ± 0.47a  4.69 ± 0.46ab  4.18 ± 0.46b     NS  0.07  NS  42 d   ADG, g  603.27 ± 23.1a  691.34 ± 23.1b  693.48 ± 23.1b  12     0.05  NS   Daily feed intake, kg  1.98  1.98  1.98               Calculated feed:gain  3.28  2.86  2.86               Backfat, mm  5.94 ± 0.68a  4.96 ± 0.68a  3.92 ± 0.68a        NS  NS   Loin-eye area, cm2  51.35 ± 2.41a  62.49 ± 2.39b  61.35 ± 2.40b        0.02  NS  Item  Control, 0 mg/kg  20 mg/kg  60 mg/kg  Pigs, n  Group  Treatment  Group × treatment  14 d   ADG, g  732.1 ± 23.0a,A  826.4 ± 23.0b,B  900 ± 24.0c,B  35  NS3  0.001  NS   Daily feed intake, kg  1.93  1.93  1.93               Calculated feed:gain2  2.60  2.34  2.14              28 d   ADG, g  630.67 ± 22.0A  738.61 ± 22.0B  757.95 ± 20.0B  24  NS  0.001  NS   Daily feed intake, kg  2.00  2.00  2.00               Calculated feed:gain  3.20  2.70  2.63               Backfat, mm  5.66 ± 0.47a  4.69 ± 0.46ab  4.18 ± 0.46b     NS  0.07  NS  42 d   ADG, g  603.27 ± 23.1a  691.34 ± 23.1b  693.48 ± 23.1b  12     0.05  NS   Daily feed intake, kg  1.98  1.98  1.98               Calculated feed:gain  3.28  2.86  2.86               Backfat, mm  5.94 ± 0.68a  4.96 ± 0.68a  3.92 ± 0.68a        NS  NS   Loin-eye area, cm2  51.35 ± 2.41a  62.49 ± 2.39b  61.35 ± 2.40b        0.02  NS  a–cWithin a row, means not sharing common superscript are different at P < 0.05. A,BWithin a row, means not sharing common superscript are different at P < 0.01. 1Yorkshire × Large White Landrace maternal line. 2Calculated feed efficiencies (feed:gain) parallel the ADG because fed intakes were identical per day for each pig in the 14-, 28-, and 42-d groups. 3NS = not significant. View Large Table 2. Performance and carcass data for pigs fed 0, 20, and 60 mg/kg of ractopamine1 Item  Control, 0 mg/kg  20 mg/kg  60 mg/kg  Pigs, n  Group  Treatment  Group × treatment  14 d   ADG, g  732.1 ± 23.0a,A  826.4 ± 23.0b,B  900 ± 24.0c,B  35  NS3  0.001  NS   Daily feed intake, kg  1.93  1.93  1.93               Calculated feed:gain2  2.60  2.34  2.14              28 d   ADG, g  630.67 ± 22.0A  738.61 ± 22.0B  757.95 ± 20.0B  24  NS  0.001  NS   Daily feed intake, kg  2.00  2.00  2.00               Calculated feed:gain  3.20  2.70  2.63               Backfat, mm  5.66 ± 0.47a  4.69 ± 0.46ab  4.18 ± 0.46b     NS  0.07  NS  42 d   ADG, g  603.27 ± 23.1a  691.34 ± 23.1b  693.48 ± 23.1b  12     0.05  NS   Daily feed intake, kg  1.98  1.98  1.98               Calculated feed:gain  3.28  2.86  2.86               Backfat, mm  5.94 ± 0.68a  4.96 ± 0.68a  3.92 ± 0.68a        NS  NS   Loin-eye area, cm2  51.35 ± 2.41a  62.49 ± 2.39b  61.35 ± 2.40b        0.02  NS  Item  Control, 0 mg/kg  20 mg/kg  60 mg/kg  Pigs, n  Group  Treatment  Group × treatment  14 d   ADG, g  732.1 ± 23.0a,A  826.4 ± 23.0b,B  900 ± 24.0c,B  35  NS3  0.001  NS   Daily feed intake, kg  1.93  1.93  1.93               Calculated feed:gain2  2.60  2.34  2.14              28 d   ADG, g  630.67 ± 22.0A  738.61 ± 22.0B  757.95 ± 20.0B  24  NS  0.001  NS   Daily feed intake, kg  2.00  2.00  2.00               Calculated feed:gain  3.20  2.70  2.63               Backfat, mm  5.66 ± 0.47a  4.69 ± 0.46ab  4.18 ± 0.46b     NS  0.07  NS  42 d   ADG, g  603.27 ± 23.1a  691.34 ± 23.1b  693.48 ± 23.1b  12     0.05  NS   Daily feed intake, kg  1.98  1.98  1.98               Calculated feed:gain  3.28  2.86  2.86               Backfat, mm  5.94 ± 0.68a  4.96 ± 0.68a  3.92 ± 0.68a        NS  NS   Loin-eye area, cm2  51.35 ± 2.41a  62.49 ± 2.39b  61.35 ± 2.40b        0.02  NS  a–cWithin a row, means not sharing common superscript are different at P < 0.05. A,BWithin a row, means not sharing common superscript are different at P < 0.01. 1Yorkshire × Large White Landrace maternal line. 2Calculated feed efficiencies (feed:gain) parallel the ADG because fed intakes were identical per day for each pig in the 14-, 28-, and 42-d groups. 3NS = not significant. View Large Northern Analysis of mRNA Abundance of the SREBP-1, FAS, GLUT4, SCD, PPARα, and PPARγ2 Genes in Porcine Adipose Tissue Summarized results (statistical comparisons of the 0, 20, and 60 mg/kg of RAC feeding at 14, 28, and 42 d) are presented in Figures 1 and 2, with representative Northern blots provided in the supplemental material (http://jas.fass.org/content/vol89/issue4/). On d 14, feeding RAC reduced mRNA abundances of FAS and GLUT4 (P < 0.05) for the 60 mg/kg of RAC treatment, but not the 20 mg/kg of RAC treatment (Figure 2), whereas the effect of RAC on SREBP-1 on d 14 was not clear because of partial degradation of RNA in the blots. By d 28, mRNA abundances for SREBP-1, FAS, and GLUT4 were decreased for 20 and 60 mg/kg of RAC (Figures 1 and 2). Not only did FAS mRNA abundance decline at d 28 compared with d 14, but FAS mRNA abundances for 20 and 60 mg/kg of RAC were similar. On d 28 and 42 for SREBP-1 and on d 28 for GLUT4, gene expression attenuation was less (P < 0.01) for 20 mg/kg of RAC than for 60 mg/kg of RAC (Figures 1 and 2). On d 42, the expression responses by FAS and SREBP-1 mirrored the expression responses for d 28 (Figures 1 and 2). The blots of GLUT4 mRNA abundance for d 42 were not acceptable for analysis (mRNA degradation) and were not repeated. In the present study, 20 and 60 mg/kg of RAC did not affect mRNA abundances of SCD at d 14, 28, and 42; however, on d 28, at 60 mg/kg of RAC, the attenuation of SCD expression approached significance (P < 0.13; Figure 2). Overall expression (mRNA abundance) of PPARα (a TF that regulates lipid oxidation) was increased (P < 0.01) for both treatments at d 14, 28, and 42. Expression in pigs fed 60 mg/kg of RAC was greater than for pigs fed 20 mg/kg of RAC (P < 0.01, d 14 and 42; P < 0.05, d 28; Figure 1). Expression (mRNA abundance) was not affected on d 14 for PPARγ2, but thereafter, PPARγ2 expression was decreased by RAC for both treatments. The PPARγ2 mRNA abundances were less at 60 mg/kg than at 20 mg/kg of RAC, and both were less than for the controls (Figure 1). Figure 1. View largeDownload slide Effect of feeding 0, 20, and 60 mg/kg of ractopamine hydrochloride to pigs on mRNA abundance in subcutaneous adipose tissue for sterol regulatory binding protein (SREBP-1; panel A; 4.2 kb), PPARα (panel B; 8.9 kb), and PPARγ2 (panel C; 1.9 kb). Tissue RNA was extracted and Northern blotting was conducted as described in the Materials and Methods section. Actual blots are presented in the supplemental material (http://jas.fass.org/content/vol89/issue4/). Expression data (normalized using β-actin as the reference gene; referred to as relative expression) are plotted for each sampling time for all 3 ractopamine concentrations (0, 20, and 60 mg/kg; n = 3). Columns not sharing a common lowercase letter (a–c, within a gene and day category) were significantly different at P < 0.05. Figure 1. View largeDownload slide Effect of feeding 0, 20, and 60 mg/kg of ractopamine hydrochloride to pigs on mRNA abundance in subcutaneous adipose tissue for sterol regulatory binding protein (SREBP-1; panel A; 4.2 kb), PPARα (panel B; 8.9 kb), and PPARγ2 (panel C; 1.9 kb). Tissue RNA was extracted and Northern blotting was conducted as described in the Materials and Methods section. Actual blots are presented in the supplemental material (http://jas.fass.org/content/vol89/issue4/). Expression data (normalized using β-actin as the reference gene; referred to as relative expression) are plotted for each sampling time for all 3 ractopamine concentrations (0, 20, and 60 mg/kg; n = 3). Columns not sharing a common lowercase letter (a–c, within a gene and day category) were significantly different at P < 0.05. Figure 2. View largeDownload slide Effect of feeding 0, 20, and 60 mg/kg of ractopamine hydrochloride to pigs on mRNA abundance in subcutaneous adipose tissue for fatty acid synthase (FAS; panel A; 10.9 kb), stearoyl-CoA desaturase (SCD; panel B; 4.9 kb), and glucose transporter 4 (GLUT4; panel C; 3.4 kb). Tissue RNA was extracted and Northern blotting was conducted as described in the Materials and Methods section. Actual blots are presented in the supplemental material (http://jas.fass.org/content/vol89/issue4/). Expression data (normalized using β-actin as the reference gene; referred to as relative expression) are plotted for each sampling time for all 3 ractopamine concentrations (0, 20, and 60 mg/kg; n = 3). Columns not sharing a common lowercase letter (a–c, within a gene and day category) were significantly different at P < 0.05. Figure 2. View largeDownload slide Effect of feeding 0, 20, and 60 mg/kg of ractopamine hydrochloride to pigs on mRNA abundance in subcutaneous adipose tissue for fatty acid synthase (FAS; panel A; 10.9 kb), stearoyl-CoA desaturase (SCD; panel B; 4.9 kb), and glucose transporter 4 (GLUT4; panel C; 3.4 kb). Tissue RNA was extracted and Northern blotting was conducted as described in the Materials and Methods section. Actual blots are presented in the supplemental material (http://jas.fass.org/content/vol89/issue4/). Expression data (normalized using β-actin as the reference gene; referred to as relative expression) are plotted for each sampling time for all 3 ractopamine concentrations (0, 20, and 60 mg/kg; n = 3). Columns not sharing a common lowercase letter (a–c, within a gene and day category) were significantly different at P < 0.05. DISCUSSION We have conducted a series of experiments on the effect of RAC on expression of lipogenic genes and related TF. The initial work used enzyme activity assays to study the effect of RAC on porcine WAT lipid metabolism (Merkel et al., 1987). The study by Reiter et al. (2007) was designed to assess the role of porcine genotypes (Duroc vs. Pietrains) on lipogenic gene expression and on differential effects of RAC on lipogenic gene expression in skeletal muscle and adipose tissues in these 2 genotypes after a complete finishing period. The present experiment was designed specifically to study lipogenic gene expression patterns in WAT of Large White Landrace pigs at a constant daily energy intake, over a 42-d feeding period, with tissue samplings at 14, 28, and 42 d. Expression of lipogenic enzymes and regulatory TF is sensitive to variations in dietary energy consumption (Sul and Wang, 1998). In this study, any effects of feed intake were negated by providing pigs the same amount of feed, irrespective of RAC treatment. All feed was consumed by the pigs and weigh-backs were not required. Hence, the observed effects of RAC on gene expression, performance, and carcass characteristics cannot be attributed to feed intake differences among treatment groups. In the present study, with pigs consuming identical amounts of DM for the 0, 20, and 60 mg/kg of RAC diets, respectively, daily BW gains were enhanced in the 20 and 60 mg/kg of RAC experimental groups and calculated feed efficiency was improved by feeding RAC. The expected positive effect of RAC on lean deposition was evident (Bergen et al., 1989; Mersmann, 1998; Armstrong et al., 2004; Mimbs et al., 2005) from the increased loin eye area after 42 d. The pigs used here were lean, exhibiting only 4 to 6 mm of measurable backfat thickness at slaughter, and backfat measures were unaffected by RAC. In this study, RAC changed mRNA abundance of both regulatory genes and genes of enzymes involved in fatty acid synthesis in porcine WAT, but these changes appeared to be dependent on both dietary RAC content and length of treatment. Here, mRNA abundance of SREBP-1 was apparently not affected by RAC on d 14 (the data were marginal because of RNA degradation in the blots and are not given) but was decreased at d 28 and 42. Likewise, the expression of PPARγ2, an important regulatory gene for adipogenesis and enhanced lipid storage (Otto and Lane, 2005), was not affected by RAC treatment after 2 wk. Thereafter, for d 28 and 42, RAC decreased PPARγ2 expression for both RAC treatments. The 60 mg/kg of RAC treatment (which is approximately 7 times greater than the recommended RAC concentrations in commercial swine feeding) resulted in less PPARγ2 expression beyond that noted for 20 mg/kg of RAC. Overall, the responses of SREBP-1 and PPARγ2 to RAC feeding were remarkably similar in that expressions of these TF were not affected on d 14, but continued chronic RAC feeding resulted in reduced expression of these TF at d 28 and 42. The present 42-d results are in agreement with our previous work in pigs in which we reported less WAT SREBP-1 and FAS expression after 52 d of RAC feeding (Reiter et al., 2007). However, Liu et al. (1994) reported that mRNA abundance of acetyl CoA carboxylase (a lipogenic enzyme) was not markedly changed in WAT by feeding RAC to pigs up to 24 d. In the present study, PPARα expression was increased with RAC treatment. This TF is associated with oxidative capacity of a tissue (Desvergne et al., 2006). The increase in PPARα expression and marked attenuation of PPARγ2 expression and expression of other lipogenic genes may, in part, reflect the “repartitioning” effect of RAC on adipose fat deposition. Fatty acid synthase mRNA abundance was decreased on d 14 (60 mg/kg only), d 28, and d 42 (20 and 60 mg/kg); abundance of GLUT4 mRNA was decreased at d 14 (60 mg/kg only) and d 28 (20 and 60 mg/kg), whereas SCD expression was only slightly affected at d 28. Sterol regulatory binding protein-1c is a major regulatory determinant of tissue differential lipogenic capacity in mammalian and avian species (Gondret et al., 2001; Horton et al., 2002; Shimano, 2002). In this work, we could demonstrate a decreased abundance for SREBP-1 only for d 28 and 42; hence, in pigs fed RAC, decreased expression of FAS on d 14 without parallel changes in SREBP-1c mRNA abundance could not be clearly evaluated. However, for d 28 and 42, RAC decreased the mRNA abundance of both FAS and SREBP-1 in WAT. These results support other work on parallel expression of SREBP-1 and FAS in porcine adipose tissue (Gondret et al., 2001; Reiter et al., 2007) and agree with the work in rodents (Osborne, 2000; Horton et al., 2002). Evidence also exists that mRNA abundance of SREBP-1c does not always coincide with insulin-mediated changes in lipogenic gene expression in adipose tissues or primary adipocytes (Palmer et al., 2002; Bertile and Raclot, 2004). Recently, the direct effect of SREBP-1c on adipocyte FAS promoter function was assessed, and the results showed that expression of the lipogenic genes was apparently controlled independently of SREBP-1c in rodent adipocytes (Sekiya et al., 2007). Ractopamine may have a direct effect on the regulation of DNL in pigs. Catecholamines (or BAA) and glucagon stimulate the synthesis of cAMP (Sul and Wang, 1998; Lefkowitz, 2006). When hepatocytes are incubated with glucagon or dibutyryl-cAMP, expression of SREBP-1 and its lipogenic target genes is reduced (Foretz et al., 1999; Yellaturu et al., 2005). We showed previously that in adipogenic TA1 cells, RAC decreased mRNA abundance of lipogenic genes in a propranolol (a BAA antagonist)-reversible manner (Dickerson, 1990; Bergen, 2001). Moreover, cAMP appeared directly involved, because nonmetabolizable cAMP analogs also attenuated lipogenic gene expression in TA1 cells in culture (Dickerson, 1990; Bergen, 2001). Expression of SREBP-1c was not measured in those TA1 cell experiments. Other workers have reported that both hepatic FAS and SREBP-1c expression were decreased directly by cAMP (Oskouian et al., 1996; Rangan et al., 1996), and nuclear factor Y has been identified as a possible promoter mediator for cAMP-induced suppression of FAS expression in liver (Roder et al., 2000; Schweizer et al., 2002; Griffin and Sul, 2004; Sul and Smith, 2008). Clarification of the role of SREBP-1c in regulating adipose lipogenic gene expression in pigs fed RAC awaits further studies. From the present observations, the mechanism whereby FAS mRNA abundance is decreased in pigs fed RAC cannot be clearly ascertained. Decreased FAS mRNA abundance may be a consequence of both a direct RAC via cAMP effect on FAS expression and SREBP-1-dependent (after 14 d) regulation of FAS expression in pig adipose tissues. Clearly, these changes in FAS, SREBP-c, and PPARγ2 expression cannot be attributed to differences in feed intake between the 0, 20, and 60 mg/kg of RAC treatments. Feeding RAC decreased GLUT4 (insulin-dependent adipose glucose transporter) mRNA abundance in porcine adipose tissue in concert with cAMP suppression of GLUT4 expression, as shown previously (Kaestner et al., 1991; Cooke and Lane, 1999; Yu et al., 2001; Qi et al., 2009). The mechanism whereby cAMP may modulate GLUT4 expression is not resolved; however, GLUT4 does not possess a cAMP response element consensus sequence in the experimentally determined cAMP response region in the proximal GLUT4 promoter, and the cAMP effect was suggested to be mediated by TF NF1 (Cooke and Lane, 1999). Stearoyl CoA desaturase has long been regarded as a key lipogenic enzyme, particularly in the liver (Flowers and Ntambi, 2008). This enzyme catalyzes the desaturation of palmitic and stearic acids, arising from de novo synthesis or from the diet, to MUFA (Flowers and Ntambi, 2008). In the past, this desaturation activity has been thought to have a primary role in maintaining fluidity of membrane lipid bilayers (Nakamura and Nare, 2004), but more recent studies have also implicated SCD in the development of obesity (Flowers and Ntambi, 2009). In rodents, VLDL synthesis occurs in the liver when using hepatic triacylglycerol. Here again, sizeable desaturation of triacylglycerol fatty acids will result in a more pliable fat that can be transferred to apolipoprotein B, then forming VLDL (Miyazaki and Ntambi, 2008). We had anticipated that in porcine adipose tissue, SCD expression may be attenuated by RAC parallel with FAS and GLUT4 expression, but in the Northern blot study, only a slight attenuation (group 2) was noted for SCD in pigs fed RAC. The Δ9-desaturation of fatty acids in porcine WAT depot lipids may not be as critical in pigs as hepatic Δ9-desaturation of fatty acids in rodents before hepatic VLDL synthesis. The present work thus reveals that at sufficient intakes (20 and 60 mg/kg) of RAC, a decreased expression of specific target genes in lipid metabolism occurs in porcine adipose tissue, particularly the genes involved in DNL and adipogenesis over a 42-d period. Our present study could not corroborate other work (Liu et al., 1994) but agreed with our previous studies (Merkel et al., 1987; Reiter et al., 2007). We observed decreased expression responses in specific target genes despite the likelihood that RAC-BAR binding was downregulated by 40 to 50% in porcine adipose tissue (Liang and Mills, 2002). Recently, Rikard-Bell et al. (2009) used RAC in a pig production trial. Ractopamine was administered in a step-up program to immunocastrated and intact boars and gilts for a 31-d feeding period. Those workers noted positive effects of RAC on final BW and ADG. Furthermore, RAC increased carcass lean content and decreased fat content, particularly in the immunocastrated and intact boars, indicating a positive effect of RAC up to 31 d. Lipogenic genes are extremely sensitive to energy status (Sul and Wang, 1998). Before slaughter, however, food animals are usually placed for several hours in a holding pen where feed is not available. Thus, tissue samples obtained after slaughter may not accurately reflect the patterns of gene expression and enzyme activity [particularly for lipogenic enzymes in porcine adipose tissue; R. A. Merkel (Michigan State University, East Lansing) and W. G. Bergen, unpublished data]. In the present study, pigs were exsanguinated without any intervening period of feed deprivation. It is not clear from the report of Liu et al. (1994) whether pigs underwent any feed deprivation before slaughter; however, their results showed that RAC had no effect on adipose acetyl-CoA carboxylase and malic enzyme activities throughout the experimental period (pretreatment, 1, 8, and 24 d). Furthermore, mRNA abundances of adipose acetyl-CoA carboxylase were not different between any of the sampling time points and treatments (pretreatment, RAC for 1, 8, and 24 d). In retrospect, the data of Liu et al. (1994) likely do not support the conclusion that RAC has, at best, only a short-term effect on lipogenesis (DNL) in porcine adipose tissues. Further work will be necessary to clarify differences between the present work and previous reports. LITERATURE CITED Armstrong T. A. Ivers D. J. Wagner J. R. Anderson D. B. Weldon W. C. Berg E. P. 2004. The effect of dietary ractopamine concentration and duration of feeding on growth performance, carcass characteristics, and meat quality of finishing pigs. J. Anim. Sci.  82: 3245– 3253. [PubMed] Google Scholar CrossRef Search ADS PubMed  Bergen W. G. 2001. The role of cAMP elevating agents and somatotropin on pre- and posttranslational regulation of lipogenesis and lipolysis in Bos taurus and Sus scrofa. Recent Res. Dev. Lipids  5: 47– 59. Bergen W. G. Johnson S. E. Skjaerlund D. M. Babbiker A. S. Ames N. K. Merkel R. A. Anderson D. B. 1989. Muscle protein metabolism in finishing pigs fed ractopamine. J. Anim. Sci.  67: 2255– 2262. [PubMed] Google Scholar CrossRef Search ADS PubMed  Bergen W. G. Mersmann H. J. 2005. Comparative aspects of lipid metabolism: Impact on contemporary research and use of animal models. J. Nutr.  135: 2499– 2502. [PubMed] Google Scholar CrossRef Search ADS PubMed  Bertile F. Raclot T. 2004. Messenger RNA levels of SREBP-1c do not coincide with changes in adipose lipogenic gene expression. Biochem. Biophys. Res. Commun.  325: 827– 834. [PubMed] Google Scholar CrossRef Search ADS PubMed  Cooke D. W. Lane M. D. 1999. Transcription factor NF1 mediates repression of the GLUT4 promoter by cyclic-AMP. Biochem. Biophys. Res. Commun.  260: 600– 604. [PubMed] Google Scholar CrossRef Search ADS PubMed  Desvergne B. Michalik L. Wahli W. 2006. Transcriptional regulation of metabolism. Physiol. Rev.  86: 465– 514. [PubMed] Google Scholar CrossRef Search ADS PubMed  Dickerson, P. S. 1990. The effect of the phenethanolamine ractopamine on lipid metabolism of the adipogenic cell line TA1. PhD Diss.  Michigan State University, East Lansing. Dickerson-Weber P. S. Merkel R. A. Bergen W. G. 1992. Adipogenic cell line TA1: A suitable model to study the effect of β-adrenergic agonists on lipid metabolism. Proc. Exp. Biol. Med.  201: 47– 53. Google Scholar CrossRef Search ADS   Ding S. T. Schinkel A. P. Weber T. E. Mersmann H. J. 2000. Expression of porcine transcription factors and genes related to fatty acid metabolism in different tissues and genetic populations. J. Anim. Sci.  78: 2127– 2134. [PubMed] Google Scholar CrossRef Search ADS PubMed  Dunshea F. R. King R. H. Campbell R. G. Sainz R. D. Kim T. Y. 1993. Interrelationship between sex and ractopamine on protein and lipid deposition in rapidly growing pigs. J. Anim. Sci.  71: 2919– 2930. [PubMed] Google Scholar CrossRef Search ADS PubMed  Dunshea F. R. King R. H. Eason P. J. Campbell R. G. 1998. Interrelationships between dietary ractopamine, energy intake and sex in pigs. Aust. J. Agric. Res.  49: 565– 574. Google Scholar CrossRef Search ADS   Flowers M. T. Ntambi J. M. 2008. Role of stearoyl Co-A desaturase in regulating lipid metabolism. Curr. Opin. Lipidol.  19: 248– 256. [PubMed] Google Scholar CrossRef Search ADS PubMed  Flowers M. T. Ntambi J. M. 2009. Stearoyl-CoA desaturase and its relation to high carbohydrate diets and obesity. Biochim. Biophys. Acta  1791: 85– 91. [PubMed] Google Scholar CrossRef Search ADS PubMed  Foretz M. Pacot C. Dugail I. Lemarchand P. Guichard C. Le Lièpvre X. Berthelier-Lubrano C. Spiegelman B. Kim J. B. Ferré P. Foufelle F. 1999. ADD1/SREBP-1c is required in the activation of hepatic lipogenic gene expression by glucose. Mol. Cell. Biol.  19: 3760– 3768. [PubMed] Google Scholar CrossRef Search ADS PubMed  Gondret F. Ferre P. Dugail I. 2001. ADD-1/SREBP-1 is a major determinant of tissue differential lipogenic capacity in mammalian and avian species. J. Lipid Res.  42: 106– 113. [PubMed] Google Scholar PubMed  Griffin M. J. Sul H. S. 2004. Insulin regulation of fatty acid synthase gene transcription: Roles of USF and SREBP-1c. IUBMB Life  56: 595– 600. [PubMed] Google Scholar CrossRef Search ADS PubMed  Hausman G. J. Dodson M. V. Ajuwon K. Azain M. Barnes K. M. Guan L. L. Jiang Z. Poulos S. P. Sainz R. D. Smith S. Spurlock M. Novakofski J. Fernyhough M. E. Bergen W. G. 2009. Board-invited review: The biology and regulation of preadipocytes and adipocytes in meat animals. J. Anim. Sci.  87: 1218– 1246. [PubMed] Google Scholar CrossRef Search ADS PubMed  Horton J. D. 2002. Sterol regulatory element-binding proteins: Transcriptional regulators of lipid synthesis. Biochem. Soc. Trans.  30: 1091– 1095. [PubMed] Google Scholar CrossRef Search ADS PubMed  Horton J. D. Goldstein J. L. Brown M. S. 2002. SREBPs: Activators of the complete program of cholesterol and fatty acid synthesis in the liver. J. Clin. Invest.  109: 1125– 1131. [PubMed] Google Scholar CrossRef Search ADS PubMed  Kaestner K. H. Flores-Riveros J. R. McLenithan J. C. Janicot M. Lane M. D. 1991. Transcriptional repression of the mouse insulin-responsive glucose transporter (GLUT4) gene by cAMP. Proc. Natl. Acad. Sci. USA  88: 1933– 1937. [PubMed] Google Scholar CrossRef Search ADS   Lefkowitz R. J. 2007. Seven transmembrane receptors: A brief personal retrospective. Biochim. Biophys. Acta  1768: 748– 755. [PubMed] Google Scholar CrossRef Search ADS PubMed  Liang W. Mills S. E. 2002. Quantitative analysis of β-adrenergic receptor subtypes in pig tissues. J. Anim. Sci.  80: 963– 970. [PubMed] Google Scholar CrossRef Search ADS PubMed  Liu C. Y. Grant A. L. Kim K. H. Ji S. Q. Hancock D. L. Anderson D. B. Mills S. E. 1994. Limitations of ractopamine to affect adipose tissue metabolism in swine. J. Anim. Sci.  72: 62– 67. [PubMed] Google Scholar CrossRef Search ADS PubMed  Merkel R. A. Dickerson P. S. Johnson S. E. Burkett R. L. Burnett R. J. Schroeder A. L. Bergen W. G. Anderson D. B. 1987. The effect of ractopamine on lipid metabolism in pigs. Fed. Proc.  46: 1177A. (Abstr.) Mersmann H. J. 1998. Overview of the effects of β-adrenergic receptor agonists on animals including mechanisms of action. J. Anim. Sci.  76: 160– 172. [PubMed] Google Scholar CrossRef Search ADS PubMed  Mimbs K. J. Pringle T. D. Azain M. J. Meers S. A. Armstrong T. A. 2005. Effects of ractopamine on performance and composition of pigs phenotypically sorted into fat and lean groups. J. Anim. Sci.  83: 1361– 1369. [PubMed] Google Scholar CrossRef Search ADS PubMed  Miyazaki, M., and J. M. Ntambi 2008. Fatty acid desaturation and chain elongation in mammals. Pages 191–211 in Biochemistry of Lipids, Lipoproteins and Membranes.  5th ed. D. E. Vance and J. E. Vance ed. Elsevier, Amsterdam, the Netherlands. Google Scholar CrossRef Search ADS   Nakamura M. T. Nare T. Y. 2004. Structure, function, and dietary regulation of Δ6, Δ5 and Δ9 desaturases. Annu. Rev. Nutr.  24: 345– 376. [PubMed] Google Scholar CrossRef Search ADS PubMed  NRC 1999. Nutrient Requirements of Swine.  10th rev. ed. Natl. Acad. Press, Washington DC. Osborne T. F. 2000. Sterol regulatory element-binding proteins (SREBPs): Key regulators of nutritional homeostasis and insulin action. J. Biol. Chem.  275: 32379– 32382. [PubMed] Google Scholar CrossRef Search ADS PubMed  Oskouian B. Rangan V. S. Smith S. 1996. Transcriptional regulation of the rat fatty acid synthase gene: Identification of functional analysis of positive and negative effectors of basal transcription. Biochem. J.  317: 257– 265. [PubMed] Google Scholar CrossRef Search ADS PubMed  Otto T. C. Lane M. D. 2005. Adipose development: From stem cell to adipocyte. Crit. Rev. Biochem. Mol. Biol.  40: 229– 242. [PubMed] Google Scholar CrossRef Search ADS PubMed  Palmer D. G. Rutter G. A. Tavaré J. M. 2002. Insulin-stimulated fatty acid synthase gene expression does not require increased sterol response element binding protein 1 transcription in primary adipocytes. Biochem. Biophys. Res. Commun.  291: 439– 443. [PubMed] Google Scholar CrossRef Search ADS PubMed  Peterla T. A. Scanes C. G. 1990. Effect of β-adrenergic agonists on lipolysis and lipogenesis by porcine adipose tissue in vitro. J. Anim. Sci.  68: 1024– 1029. [PubMed] Google Scholar CrossRef Search ADS PubMed  Qi L. Saberi M. Zmuda E. Wang Y. Altarejos J. Zhang X. Dentin R. Hedrick S. Bandyopadhyay G. Hai T. Olefsky J. Montminy M. 2009. Adipocyte CREB promotes insulin resistance in obesity. Cell Metab.  9: 277– 286. [PubMed] Google Scholar CrossRef Search ADS PubMed  Rangan V. S. Oskouian B. Smith S. 1996. Identification of an inverted CCAAT box motif in the fatty acid synthase gene as an essential element for mediation of transcriptional regulation by cAMP. J. Biol. Chem.  271: 2307– 2312. [PubMed] Google Scholar CrossRef Search ADS PubMed  Reiter S. S. Halsey C. H. C. Stronach B. J. Bartosh J. L. Owsley W. F. Bergen W. G. 2007. Lipid metabolism related gene-expression profiling in liver, skeletal muscle and adipose tissue in crossbred Duroc and Pietrain pigs. Comp. Biochem. Physiol. D Genomics Proteomics  2: 200– 206. [PubMed] Google Scholar CrossRef Search ADS   Rikard-Bell C. Curtis M. A. van Barneveld R. J. Mullan B. P. Edwards A. C. Gannon N. J. Henman D. J. Hughes P. E. Dunshea F. R. 2009. Ractopamine hydrochloride improves growth performance and carcass composition in immunocastrated boars, intact boars, and gilts. J. Anim. Sci.  87: 3536– 3543. [PubMed] Google Scholar CrossRef Search ADS PubMed  Roder K. Wolf S. S. Schweizer M. E. 2000. Dependence of rat spot 14 promoter activity on NF-Y binding to the inverted CCAAT-element at −100. Biosci. Biotechnol. Biochem.  64: 848– 851. [PubMed] Google Scholar CrossRef Search ADS PubMed  Schweizer M. E. Roder K. Zhang L. Wolf S. S. 2002. Transcription factors acting on the promoter of rat fatty acid synthase gene. Biochem. Soc. Trans.  30: 1070– 1072. [PubMed] Google Scholar CrossRef Search ADS PubMed  Sekiya M. Yahagi N. Matsuzaka T. Takeuchi Y. Nakagawa Y. Takahashi H. Okazaki H. Iizuka Y. Ohashi K. Gotoda T. Ishibashi S. Nagai R. Yamazaki T. Kadowaki T. Yamada N. Osuga J. Shimano H. 2007. SREBP-1 independent regulation of lipogenic gene expression in adipocytes. J. Lipid Res.  48: 1581– 1591. [PubMed] Google Scholar CrossRef Search ADS PubMed  Shimano H. 2002. Sterol regulatory element-binding protein family as global regulators of lipid synthetic genes in energy metabolism. Vitam. Horm.  65: 167– 194. [PubMed] Google Scholar CrossRef Search ADS PubMed  Shimomura I. Shimano H. Horton J. D. Goldstein J. L. Brown M. S. 1997. Differential expression of exons 1a and 1c for sterol regulatory element binding protein-1 in human and mouse organs and cultured cells. J. Clin. Invest.  99: 838– 845. [PubMed] Google Scholar CrossRef Search ADS PubMed  Sillence M. N. 2004. Technologies for the control of fat and lean deposition in livestock. Vet. J.  167: 242– 257. [PubMed] Google Scholar CrossRef Search ADS PubMed  Sul, H. S., and S. Smith 2008. Fatty acid synthesis in eukaryotes. Pages 155–190 in Biochemistry of Lipids, Lipoproteins and Membranes.  5th ed. D. E. Vance and J. E. Vance ed. Elsevier, Amsterdam, the Netherlands. Google Scholar CrossRef Search ADS   Sul H. S. Wang D. 1998. Nutritional and hormonal regulation of enzymes in fat synthesis: Studies of fatty acid and mitochondrial glycerol-3-phosphate acyltransferase gene transcription. Annu. Rev. Nutr.  18: 331– 351. [PubMed] Google Scholar CrossRef Search ADS PubMed  Trayhurn P. 1996. Northern blotting. Proc. Nutr. Soc.  55: 583– 589. [PubMed] Google Scholar CrossRef Search ADS PubMed  Yellaturu C. R. Deng X. Cagen L. M. Wilcox H. G. Park E. A. Raghow R. Elam M. B. 2005. Posttranslational processing of SREBP-1 in rat hepatocytes is regulated by insulin and cAMP. Biochem. Biophys. Res. Commun.  332: 174– 180. [PubMed] Google Scholar CrossRef Search ADS PubMed  Yu Z. W. Buren J. Enerback S. Nilsson E. Samuelsson L. Eriksson J. W. 2001. Insulin can enhance GLUT4 gene expression in 3T3-F442A cells and this effect is mimicked by vanadate but counteracted by cAMP and high glucose: Potential implications for insulin resistance. Biochim. Biophys. Acta  1535: 174– 185. [PubMed] Google Scholar CrossRef Search ADS PubMed  Footnotes 1 The work presented herein was supported by the Alabama Agricultural Experiment Station (Auburn), the Michigan Agricultural Experiment Station (Mt. Pleasant), Lilly Research Laboratories (Greenfield, IN), the Upchurch Fund for Excellence in Animal Sciences at Auburn University (Auburn), the Alabama Pork Producers Association (Montgomery), and USDA-National Research Initiative (Washington, DC) grants 93-03336 and 2004-35206-14123 to W. G. Bergen. Special thanks go to David B. Anderson, Emerson L. Potter, and Aubrey L. Schroeder (Lilly Research Laboratories, Elanco Animal Health, Indianapolis, IN) for support during this research. A portion of this work was presented in abstract form at Experimental Biology 2003 (San Diego, CA; April 11–15, 2003). American Society of Animal Science

Journal

Journal of Animal Science – Oxford University Press

Published: Apr 1, 2011

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