New insights into polar overdominance in callipyge sheep.

The callipyge phenotype in sheep involves substantial postnatal muscle hypertrophy and other changes to carcass composition. A single nucleotide polymorphism in the DLK1-DIO3 imprinted gene cluster alters gene expression of the paternal allele-specific protein-coding genes and several maternal allele-specific long noncoding RNA and microRNA when the mutation is inherited in cis. The inheritance pattern of the callipyge phenotype is polar overdominant because muscle hypertrophy only occurs in heterozygous animals that inherit a normal maternal allele and the callipyge SNP on the paternal allele (+/C). We examined the changes of gene expression of four major transcripts from the DLK1-DIO3 cluster and four myosin isoforms during the development of muscle hypertrophy in the semimembranosus as well as in the supraspinatus that does not undergo hypertrophy. The homozygous (C/C) animals had an intermediate gene expression pattern for the paternal allele-specific genes and two myosin isoforms, indicating a biological activity that was insufficient to change muscle mass. Transcriptome analysis was conducted by RNA sequencing in the four callipyge genotypes. The data show that homozygous animals (C/C) have lower levels of gene expression at many loci relative to the other three genotypes. A number of the downregulated genes are putative targets of the maternal allele-specific microRNA with gene ontology, indicating regulatory and cell signaling functions. These results suggest that the trans-effect of the maternal noncoding RNA and associated miRNA is to stabilize the expression of a number of regulatory genes at a functional, but low level to make the myofibers of homozygous (C/C) lambs less responsive to hypertrophic stimuli of the paternal allele-specific genes.

Impact of porcine maternal aerobic exercise training during pregnancy on endothelial cell function of offspring at birth.

The purpose of this investigation was to test the hypothesis that maternal exercise training during pregnancy enhances endothelial function in offspring at birth. Six-month-old gilts (n = 8) were artificially inseminated and randomized into exercise-trained (n = 4) and sedentary groups (n = 4). Exercise training consisted of 15 weeks of treadmill exercise. The thoracic aorta of offspring were harvested within 48 h after birth and vascular responsiveness to cumulative doses of endothelium-dependent (bradykinin: 10-11-10-6 M) and independent (sodium nitroprusside: 10-10-10-4 M) vasodilators were assessed using in vitro wire myography. Female offspring from the exercised-trained gilts had a significantly greater endothelium-dependent relaxation response in the thoracic aorta when compared with the male offspring and female offspring from the sedentary gilts. The results of this investigation demonstrate for the first time that maternal exercise during pregnancy produces an enhanced endothelium-dependent vasorelaxation response in the thoracic aortas of female offspring at birth.

Alterations in the transcriptome of porcine oocytes derived from prepubertal and cyclic females is associated with developmental potential.

The developmental competence of oocytes is progressively attained as females approach puberty. The poor quality of prepubertally derived oocytes suggests that essential processes during cytoplasmic maturation have not been completed. The objective of this experiment was to identify genes in oocytes that are associated with good (cyclic females) and poor (prepubertal females) developmental competence. Development to the blastocyst stage in vitro was significantly decreased in oocytes derived from prepubertal females compared with cyclic females (5.26 and 12.86%, respectively). Approximately 10% of the oocyte transcriptome was differentially expressed between in vitro-matured oocytes derived from cyclic and prepubertal females (P < 0.05); 58% of differentially expressed genes had increased transcript abundance in oocytes derived from cyclic females. Genes involved in the metabolism and regulation of biological processes had increased transcript abundance in oocytes derived from cyclic females, whereas genes involved in translation were increased in prepubertally derived oocytes. Quantitative PCR confirmed differential expression (P < 0.05) for 6 out of 11 selected genes [DPYD (dihydropyrimidine dehydrogenase), RDH11 (retinol dehydrogenase 11), SFRS4 (serine/arginine-rich splicing factor 4), SFRS7 (serine/arginine-rich splicing factor 7), TL4 (transcribed loci 4), and TOP2B (topoisomerase II ß)] that were differentially expressed with greater than a 2-fold change by microarray, although 3 of these genes, DPYD, TL4, and TOP2B, were in opposing directions by the 2 methods. In conclusion, expression of multiple genes involved in metabolism and translation was significantly altered in oocytes from prepubertal females compared with cyclic females, which was associated with reduced in vitro development to the blastocyst stage. These genes may represent important cellular mechanisms that regulate oocyte quality.

Molecular cloning and characterization of porcine calcineurin-alpha subunit expression in skeletal muscle.

The calmodulin/Ca2+-dependent serine/threonine phophatase, calcineurin (CaN), has been implicated in controlling muscle fiber phenotype. However, little information is available concerning the expression of CaN in porcine skeletal muscle. Therefore, the porcine CaN alpha (CaN-A) was cloned by reverse transcription-PCR and its expression characterized in selected porcine skeletal muscles. We successfully cloned porcine CaN gene using semitendinosus muscle (GenBank accession number AF193515). Sequence analysis showed both the full length and a 30-bp deletion splice variant in coding region of the gene reported in other species. The deduced AA sequence showed 99.4% homology with the rat CaN-A delta isoform gene. Real-time PCR analysis showed CaN is present in all tissues. However, using primers targeting the region containing the 30-bp deletion, the full length sequence is only found in skeletal muscle and brain tissues. Using a CaN-A monoclonal antibody, we localized CaN-A in porcine LM and soleus muscle and the red and white portions of the semitendinosus muscle. The CaN-A protein was abundant in fast fibers and primarily localized in the cytoplasm, whereas slow fibers expressed reduced abundance of CaN-A. Further studies are required to understand the functions of CaN-A isoform in skeletal muscle.

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

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

Cloning the genomic sequence and identification of promoter regions of bovine pyruvate carboxylase.

Pyruvate carboxylase (PC) catalyzes a pivotal reaction in gluconeogenesis and lipid metabolism in liver. In bovine the PC gene is expressed as six 5' untranslated region (UTR) mRNA variants. The objectives for this study were to clone and sequence the bovine PC gene, determine the intron and exon organization and identify PC promoter region(s). Oligonucleotide sequences that corresponded to the 5' UTR mRNA variants and coding sequence of bovine PC were used to isolate 2 clones from the RPCI-42 bovine bacterial artificial chromosome (BAC) library. Sequencing data confirmed the presence of regions for the 5' UTR for bovine PC mRNA. The exon arrangement from 5' to 3' is 48 (exon I), 41 (exon II), 178 (exon IIIA and IIIB), and 185 (exon IV) bp. Three promoter regions, P3, P2, and P1, adjacent to exon I, II, and IIIA, respectively, were identified based on computer analysis of sequence data. Putative promoters were cloned into a firefly luciferase vector and transiently transfected into H4IIE rat hepatoma cells. All PC promoters demonstrated luciferase activity comparable with the minimal promoter luciferase vector and higher than the promoterless luciferase vector. In addition, PC promoter 1 exhibited greater luciferase activity compared with PC promoter 2 or 3. These data provide information about the arrangement of the 4 bovine PC 5' UTR exons, the identity of the promoter regions for the bovine PC gene, and indicate differences in relative basal activity of the promoter regions.

Analysis of gene expression during the onset of muscle hypertrophy in callipyge lambs.

The callipyge mutation causes postnatal muscle hypertrophy in heterozygous lambs that inherit a paternal callipyge allele (+/CLPG). Our hypothesis was that the up-regulation of one or both of the affected paternally expressed genes (DLK1 or PEG11) initiates changes in biochemical and physiological pathways in skeletal muscle to induce hypertrophy. The goal of this study was to identify changes in gene expression during the onset of muscle hypertrophy to identify the pathways that are involved in the expression of the callipyge phenotype. Gene expression was analysed in longissimus dorsi total RNA from lambs at 10, 20, and 30 days of age using the Affymetrix Bovine Expression Array. An average of 40.6% of probe sets on the array was detected in sheep muscle. Data were normalized and analysed using a two-way anova for genotype and age effects with a false discovery rate of 0.10. From the anova, 13 genes were significant for the effect of genotype and 13 were significant for effect of age (P < 0.10). No significant age-by-genotype interactions were detected (P > 0.10). Of the 13 genes indicating an effect of genotype, quantitative PCR assays were developed for all of them and tested on a larger group of animals from 10 to 200 days of age. Nine genes had significantly elevated transcript levels in callipyge lambs. These genes included phosphofructokinase, a putative methyltransferase protein, a cAMP phosphodiesterase, and the transcription factor DNTTIP1.

Postnatal changes in the expression of genes located in the callipyge region in sheep skeletal muscle.

The expression of five genes surrounding the callipyge (CLPG) mutation was analysed in skeletal muscles from lambs at one prenatal and two postnatal ages that coincide with the onset and establishment of muscle hypertrophy. Genotype-specific changes in transcript abundance were detected for paternal allele-specific DLK1 and PEG11 (the official symbol of the latter is RTL1) and the maternal allele-specific MEG3, PEG11AS and MEG8 when the mutation was inherited in cis. There were differences in the temporal and muscle-specific effects on expression between the maternal allele-specific genes and paternal allele-specific genes. Maternal inheritance of the CLPG allele had a significant effect on the expression of MEG3 and MEG8 at prenatal and postnatal ages, whereas paternal inheritance of DLK1 and PEG11 only affected postnatal expression. Genotype-specific changes in PEG11AS expression were detected only in prenatal muscle. Maternal inheritance of the mutation caused similar changes in MEG3 and MEG8 expression in the semimembranosus, which undergoes hypertrophy, and the supraspinatus, which does not hypertrophy. Paternal inheritance of the mutation caused changes in PEG11 expression in both muscles, although the magnitude of expression in semimembranosus was more than 100-fold greater than in supraspinatus. DLK1 expression was upregulated in callipyge animals at both postnatal ages in the semimembranosus, but there was no effect of genotype on DLK1 expression in the supraspinatus at any age. Increased DLK1 expression was likely the primary cause of muscle hypertrophy, but a contribution of PEG11 to the phenotype cannot be ruled out based on gene expression.

Stereoselectivity of porcine beta-adrenergic receptors for ractopamine stereoisomers.

Ractopamine HCl is a beta-adrenergic receptor ((betaAR) ligand approved for use in swine to enhance carcass leanness. Ractopamine is produced commercially as a mixture of four stereoisomers (RR, RS, SR, SS). In order to determine which stereoisomers are active in the pig and whether they exhibit betaAR subtype selectivity, receptor affinity and adenylyl cyclase activation were determined using cloned porcine beta1- and beta2AR expressed in Chinese hamster ovary (CHO) cells. Dissociation constants (Kd) were determined by competitive displacement of [125I]iodocyanopindolol binding by ractopamine stereoisomers. The RR isomer had the highest affinity for both beta1- and betaAR (Kd of 29 and 26 nM, respectively). Dissociation constants for the other stereoisomers were higher (RS = 463 and 78 nM, SR = 3,230 and 831 nM, SS = 16,600 and 3,530 nM for the beta1- and beta2AR, respectively) relative to the RR stereoisomer. Isoproterenol stimulated adenylyl cyclase activity 600% relative to basal rates in CHO cells, regardless of betaAR subtype. Ractopamine stereoisomers did not significantly (P > 0.05) stimulate adenylyl cyclase through the beta1AR at moderate (near Kd) or high (10(-4) M) concentrations. In contrast, the RR isomer increased adenylyl cyclase activity 200 to 300% relative to basal rates through the beta2AR at moderate and hiconcentrations; the SR stereoisomer increased adenylyl cyclase activity nearly 100%. Neither the RS nor SS stereoisomers were effective in activating adenylyl cyclase activity through the beta2AR. A pattern of stereoselective activation similar to that for adenylyl cyclase also was exhibited for lipolysis using porcine adipocytes. The RR stereoisomer was equal to isoproterenol in stimulating lipolysis, whereas the SR isomer was 50% as effective; the RS and SR stereoisomers did not stimulate lipolysis in porcine adipocytes. The porcine betaAR exhibited stereoselectivity toward ractopamine stereoisomers with the RR isomer exhibiting the highest affinity for the (beta1- and beta2AR. In contrast, ractopamine stereoisomers seemed to be more effective at eliciting adenosine cyclic 3',5'-phosphate responses from beta2AR than beta1AR. The RR isomer ilikely the functional stereoisomer of ractopamine, but its effectiveness may be compromised by the presence of competing isomers, in particular the RS stereoisomer.

Nutritionally induced adipose hypertrophy in young pigs is transient and independent of changes in the expression of the obese and peroxisome proliferator activated receptor genes.

Previous studies have shown that piglets weaned to a liquid milk replacer (MR), rather than a typical dry diet (DD) regimen, have improved growth rates and deposit more energy as body fat. In the present study, we used this model to determine whether changes in the expression of genes linked to the regulation of adiposity were related to the accelerated fat accretion. We also determined whether the increase in body fat was sustained throughout a substantial proportion of the growth curve. At weaning (19 plus minus 2 days of age), 96 piglets were placed in 12 replicate pens per diet (4 pigs per pen, 2 barrows and 2 gilts), and fed a liquid MR or conventional DD regimen for 5 weeks. Thereafter, 6 barrows and 6 gilts pigs from each diet were killed for determination of whole body chemical composition (less gastrointestinal contents). The remaining pigs were assigned randomly to weight target groups (60, 85, and 110 kg), placed in individual pens, and fed a conventional dietary regimen until killed at their respective weight targets for tissue sampling and determination of whole body chemical composition. Over the 5-week period in which the MR was fed, the growth rate of the pigs consuming the MR exceeded that of the pigs fed the DD by 36% (P <.05). Fat gain in these pigs was increased to 1.8 times that of the pigs fed the DD, and percentage body fat was 45% greater (P <.05). Acetyl Co-A carboxylase (ACC) activity (per mg of adipose extract protein) was not different between the two diet groups at the conclusion of the 5-week period, or at 110 kg body weight. During the MR period, actual protein gain was increased (P <.05) 22% in the pigs fed the MR as well. By 110 kg of body weight, body fat was reduced (P <.05) by 7.7% (total fat mass) and 8.3% (percentage of body weight basis) in the pigs fed MR vs. the DD group. The expression of the peroxisome proliferator activated receptors (PPAR) alpha and gamma was not influenced by diet or by body weight. Expression of the obese gene was independent of diet, but was greater (P <.09) in pigs at 110 kg body weight than at 60 kg. These data provide additional evidence that piglets weaned to liquid diets have greater rates of growth and deposit more body fat, but that this difference subsides quickly when a typical dry dietary regimen is imposed. Furthermore, the biochemical changes responsible for the increased adiposity are independent of changes in the expression of the obese or PPAR genes, at least at the mRNA level.

Differential expression of the GTL2 gene within the callipyge region of ovine chromosome 18.

The inheritance pattern of the skeletal muscle hypertrophy phenotype caused by the callipyge gene has been characterized as polar overdominance. We hypothesized that this trait may be caused by a gain or loss of gene expression because of the reversible nature of the phenotype in paternal vs. maternal inheritance. Suppression subtraction cDNA probes were made from skeletal muscle mRNA of normal (NN) and callipyge (C(Pat)N(Mat)) animals and hybridized to Southern blots containing bacterial artificial chromosomes (BACs) that comprise a physical contig of the callipyge region. The CN-NN probes hybridized to two ovine and seven bovine BACs. Sequence analysis of fragments within those BACs indicated short regions of similarity to mouse gene trap locus (gtl2). Northern blots analysis of RNA from hypertrophy-responsive muscles show a population of GTL2 mRNA centred around 2.4 kb that were abundantly expressed in 14-day prenatal NN and C(Pat)N(Mat) lambs but were down-regulated in day 14 and day 56 postnatal NN lambs. The expression of GTL2 remained elevated in 14- and 56-day-old C(Pat)N(Mat) lambs as well as in 56-day-old N(Pat)C(Mat) and CC lambs. Expression of GTL2 in the supraspinatus, which does not undergo hypertrophy, was very low for all genotypes and ages. Isolation of cDNA sequences show extensive alternative splicing and a lack of codon bias suggesting that GTL2 does not encode a protein. The mutation of the callipyge allele has altered postnatal expression of GTL2 in muscles that undergo hypertrophy and will help identify mechanisms involved in growth, genomic imprinting and polar overdominance.

Expression of the porcine beta2-adrenergic receptor in Chinese hamster ovary cells.

The gene for the porcine beta2-adrenergic receptor (pbeta2AR) was transfected into Chinese hamster ovary (CHO) cells for expression. Fourteen stable cell lines were obtained and exhibited receptor densities ranging from 12 to 2,371 fmol/mg membrane protein. The receptor density was not correlated with estimates of gene copy number obtained by Southern hybridization. The pbeta2AR in CHO cells exhibited saturable binding of [125I]CYP (Kd = 14.5 pM) and stereospecificity for (-)- and (+)-isoproterenol. The relative affinities for (-)-isoproterenol (ISO), (-)-epinephrine (EPI), and (-)-norepinephrine (NEPI) were ISO > EPI > NEPI, which are characteristic of beta2AR. The affinity values for these ligands were similar to those in other species. Binding of ISO, EPI, and NE revealed two affinity states of the betaAR; the high-affinity state was eliminated by adding Gpp(NH)p, a nonhydrolyzable GTP analogue. Binding of the antagonist propranolol modeled to only one affinity state, and Gpp(NH)p did not affect binding. Multiple affinity states are characteristic of agonist-induced coupling of betaAR with G-proteins, and the data suggest that the cloned pbetaAR is functionally competent. Data confirm that the pbeta2AR is the pig version of beta2AR. Stable CHO cell lines will be useful for characterization of pbeta2AR and screening and designing potential drugs that may be used to enhance pig production.

Regulation of PPARgamma but not obese gene expression by dietary fat supplementation.

Leptin, the product of the obese gene, and peroxisome proliferator activated receptor gamma (PPARgamma) are important regulators of energy metabolism, adipogenesis, and immune function. In rodent models, both genes seem to respond at the mRNA and/or protein levels to dietary fat consumption. To determine the effect(s) of dietary saturated and polyunsaturated fatty acids on the expression (mRNA abundance) of these genes, adipose tissue was obtained from pigs fed three different dietary fat sources. Corn-soybean meal diets containing no added fat (NO, control) or 10% beef tallow (BT), safflower oil (SO), or fish oil (FO) were fed ad libitum (n = 12) for 12 weeks. The abundance of obese, PPARgamma1, and PPARgamma2 mRNA was quantified relative to 18S rRNA using ribonuclease protection assays. The gain:feed ratio was improved (P < 0.05) 21% by all fats with a corresponding reduction (P < 0.05) in feed intake. Relative to pigs fed NO, serum total cholesterol was increased (P < 0.01) in pigs fed BT and triglyceride and nonesterified fatty acid concentrations were increased (P < 0.01) by all supplemental fats. Serum insulin was increased (P < 0.10) only by SO. Neither obese nor PPARgamma1 mRNA abundance were responsive to added fat (P > 0.15). However, the abundance of PPARgamma2 mRNA was increased fourfold by SO compared with the NO diet. These data indicate that the abundance of obese mRNA is independent of dietary fat consumption per se, whether saturated or unsaturated, when feed consumption is reduced due to greater dietary caloric density. Furthermore, we provide evidence that expression of the PPARgamma2 gene in porcine adipose tissue is selectively responsive to SO (presumably linoleic acid, 18:2n-6).

Epitope-tagged insulin-like growth factor-I expression in muscle.

Development of a recombinant insulin like growth factor I (IGF-I) that is distinguishable from its endogenous counterpart would provide a powerful tool for delineating the role of IGF in myogenesis. Therefore, the objective of this study was to create an epitope-tagged IGF-I that retains biological activity and determine whether expression of this construct is possible in muscle tissue following direct DNA injection. Expression vectors were created that encoded porcine IGF-I containing a T7 (11-amino acid) epitope-tag (TIGF). Immunoreactivity of the purified recombinant TIGF was confirmed using monoclonal antibodies. Biological activity was evaluated by examining differentiation of myoblasts cultured with TIGF or transfected with TIGF plasmid DNA. Addition of purified TIGF to myoblast cultures stimulated (P < 0.05) muscle creatine kinase levels similar to insulin (10(-5) M). Likewise, transfection of L6A1 with TIGF DNA hastened (P < 0.01) differentiation compared to control pcDNA-transfected myoblasts. The integrity of the recombinant protein was confirmed using a sandwich-configured enzyme linked immunosorbent assay. Finally, recombinant TIGF DNA was injected in porcine muscle and the ability to detect TIGF protein was evaluated. TIGF expression was detected in muscle fibers of injected porcine muscle. These data show that a T7 amino acid tag placed on the amino terminus of the IGF-I protein remains intact during processing and does not interfere with the biological activity of the molecule. Use of this DNA construct is an excellent tool for investigating the role of IGFs in control muscle development and provides a model to investigate other regulators of animal growth.

Plasmid transfection and retroviral transduction of porcine muscle cells for cell-mediated gene transfer.

Cell-mediated gene transfer is a potential tool for studying muscle growth, but efficient genetic manipulation and implantation strategies have not been developed for pigs. The objectives of the present study were to determine methods for transient and stable incorporation of reporter genes into porcine muscle cells and to investigate their use for cell-mediated gene transfer in pigs. Porcine myoblasts and fibroblasts were isolated from muscle of 2-wk-old male pigs. Myogenic cell lines were identified using muscle-specific monoclonal antibodies, myotube fusion assays, and the presence of muscle-specific markers (MyoD and desmin). Four commercial cationic liposomes (lipofectAMINE, lipofectin, cellFECTIN, and DMRIE-C) were tested at different DNA:lipid ratios for their ability to transfect myoblasts and fibroblasts transiently with a luciferase reporter plasmid. LipofectAMINE resulted in the greatest (P < .01) transient luciferase activity for both cell types. Electroporation of cells for transient transfection resulted in less luciferase activity than cationic transfection. Stable transfections were conducted using a green fluorescence protein (GFP) reporter plasmid containing the neomycin resistance gene. LipofectAMINE transfection resulted in stable GFP expression in 1:16,000 myoblasts and 1:33,000 fibroblasts. Stable electroporation resulted in efficiencies that were significantly lower than established with cationic liposomes. Porcine cells were transduced with GFP using vesicular stomatitis virus glycoprotein G pseudotyped retrovirus and resulted in efficiencies of 1:1.2 for myoblasts and 1:1.1 for fibroblasts. These results show that cationic liposomes are superior to electroporation for transfection, but retroviral transduction produced stable reporter gene expression in > 80% of porcine muscle cells. Transduced GFP-positive cells were separated from GFP-negative cells by fluorescence-activated cell sorting and implanted into 2-wk-old male pigs. On d 4, implanted muscles were removed and subjected to immunodetection of GFP protein. Fibroblast implantation resulted in limited GFP expression within muscle, whereas myoblast implantation resulted in GFP within muscle fibers. This suggests that cell-mediated gene transfer is possible in porcine muscle and may be useful as an approach for studying muscle growth in pigs.

Physiological response to acute endotoxemia in swine: effect of genotype on energy metabolites and leptin.

Certain high lean gain swine genotypes have greater sensitivity to pathogen and nonpathogen stressors evident by reduced productivity and increased mortality during disease stress or in suboptimal production environments. Saline (control) and an immunologic challenge (LPS; 25 microg lipopolysaccharide/kg BW) were administered to three genetic populations (each pig used as its own control): high lean (H), moderate lean terminal cross (MT), and moderate lean maternal cross (MM). LPS induced anorexia, and significantly increased body temperature and circulating TNF-alpha, cortisol, and NEFA in all genotypes (P < 0.0004). LPS reduced circulating glucose, insulin, and IGF-1 in all genotypes (P < 0.05). The LPS-induced hypoglycemia was significantly greater in MM versus MT and H pigs (P < 0.03). The hypoinsulinemia was significantly greater in MM versus H pigs (P < 0.02). MM pigs recovered from hypoinsulinemia slower than MT pigs (P < 0.03). Control insulin was higher in H versus MT pigs (P < 0.08), but relative to basal, the insulin response to LPS was similar. Plasma haptoglobin response to LPS was lower for MM versus MT and H pigs (P < 0.02), and tended to be lower in MT versus H pigs (P < 0.09). LPS treatment caused similar decreases in plasma IGF-1 concentrations among genotypes. Ten hours after LPS treatment, leptin mRNA abundance in adipose tissue was significantly reduced (relative to control) in MM and H pigs (P < 0.02) but not in MT pigs (P > 0.05). Physiological differences in leptin, a potent regulator of food intake and energy metabolism, may be important factors in the genetic variation in sensitivity to environmental stress.

Leptin expression is reduced with acute endotoxemia in the pig: correlation with glucose, insulin, and insulin-like growth factor-1 (IGF-1).

Leptin has been implicated in the regulation of anorexia associated with cachexia in rodents and humans. Regulation of leptin expression is under complex endocrine and metabolic control. To determine if leptin expression is regulated by acute inflammation and to define the endocrine and metabolic factor(s) that regulates leptin expression during acute inflammation, castrate male pigs (ad libitum fed, used as their own controls) were treated with saline (control period) and endotoxin (lipopolysaccharide [LPS] period). Frequent blood samples were collected to identify dynamic changes in hormones and metabolites that are known to regulate leptin expression. LPS caused fever and elevated plasma cortisol (p < 0.0004), tumor necrosis factor-alpha (TNF-alpha) (p < 0.0001), and plasma nonesterified fatty acids (NEFA) (p < 0.001) compared with control. Circulating insulin (p < 0.01), glucose (p < 0.003), and insulin-like growth factor-1 (IGF-1) (p < 0.0001), as well as adipose leptin mRNA abundance (p < 0.01), were profoundly reduced following LPS treatment compared with control. Our data indicate that during acute endotoxemia (1-10 h after injection), leptin gene expression is decreased compared with ad libitum fed animals and is more closely related to energy homeostasis than cytokine profiles in plasma.

Rapid purification of transfected porcine muscle cells.

A detailed methodology is described for fluorescence-activated cell sorting (FACS) of porcine muscle cells that have been transfected to express green fluorescent protein (GFP). Cells are liberated from porcine skeletal muscle and primary cultures are transfected with DNA encoding GFP. Primary cultures are subjected to immunocytochemistry using a primary muscle-specific monoclonal antibody followed by incubation with a phycoerythrin-conjugated second antibody. Transfected myoblasts are sorted from fibroblasts using forward angle light scatter and ninety degree light scatter, phycoerythrin fluorescence, and GFP fluorescence. These procedures allow for isolation of genetically- engineered porcine muscle cells more rapidly than traditional clonal selection procedures. Consequently, FACS provides porcine myoblast populations that retain the majority of their replicative capacity and are not contaminated with non-myogenic cells.

Isolation of two populations of myoblasts from porcine skeletal muscle.

Studies on the effects of time and passage on porcine primary muscle cell cultures and methods to purify myoblasts were conducted using flow cytometry and fluorescence-activated cell sorting (FACS). Primary muscle cells cultured on single plates revealed a small cell (<10 mm diameter) population consisting of 90% desmin-positive myoblasts and a large cell (> or = 10 mm diameter) population containing desmin-positive myoblasts and nonmyoblasts. The small myoblasts were detectable up to 28 days but after cell sorting and passage, they became indistinguishable from the large myoblast population. This indicates that pig muscle contains small self-renewing myoblasts similar to humans, that become larger when induced to proliferate. A human myoblast-specific monoclonal antibody allows FACS of both large and small myoblasts from primary cells within 2 days of culture and independent of passage. These characteristics of porcine myoblasts indicate that the pig may be a suitable large animal model for myoblast-mediated gene transfer.