Enteroendocrine peptides, growth, and the microbiome during the porcine weaning transition.

BACKGROUND: Growth rate in pigs can be affected by numerous factors that also affect feeding behavior and the microbiome. Recent studies report some communication between the microbiome and the enteroendocrine system. The present study examined if changes in the piglet microbiome between birth and during the weaning transition can be correlated either positively or negatively with growth rate and plasma concentrations of enteroendocrine peptides. RESULTS: During the post-weaning transition, a 49% reduction in average daily gain was observed at day 24 (P < 0.05) relative to day 21. Pigs recovered by day 28 with body weight and average daily gain increases of 17% and 175%, respectively relative to day 24 and the highest rate of gain was measured at day 35 (462 g/day). The time interval between day 21-24 had the highest number of correlations (n = 25) between the relative abundance differences in taxa over time and corresponding percent weight gain. Amplicon sequence variants with the greatest correlation with percent weight gain between day 21-24 belonged to families Prevotellaceae NK3B31 (ρ = 0.65, P < 0.001), Veillonellaceae (ρ = 0.63, P < 0.001) and Rikenellaceae RC9 (ρ = 0.62, P < 0.001). Seven taxa were positively correlated with percent weight gain between day 24-28. Eight taxa were positively correlated with percent weight gain between day 28-35, of which four were Clostridia. Only Lactobacillus reuteri was positively correlated across both day 24-28 and day 28-35 analyses. Insulin-like growth factor 1 (IGF-1; R2 = 0.61, P < 0.001), glucose-dependent insulinotropic polypeptide (GIP; R2 = 0.20, P < 0.001), glucagon-like peptide 1 (GLP-1; R2 = 0.51, P < 0.001), and glucagon-like peptide 2 (GLP-2; R2 = 0.21, P < 0.001) were significantly associated with the piglet fecal community NMDS, while serotonin showed no significant association (R2 = 0.03, P = 0.15). Higher concentrations of GLP-1 and GLP-2 characterized day 1 fecal communities, while GIP levels had the strongest relationship primarily with samples ordinated with the day 21 cluster. CONCLUSIONS: Demonstration of an association of certain taxa with individual gut peptides at specific ages suggests the potential for the microbiome to elicit changes in the gut enteroendocrine system during early postnatal development in the pig.

Peripheral histamine and neonatal growth performance in swine.

Identification of plasma and/or serum markers at birth that will predict animal performance may be useful for identifying animals susceptible to poor growth. Metabolomic analysis of plasma from newborn swine was used to identified potential metabolite differences between 8 pairs of littermates with similar birth weights but whose ADG differed by >50 g/d so that, at weaning (21 d), littermates differed in BW by 1.62 kg (P < 0.01). Plasma analysis failed to identify metabolic pathways impacted by growth, most likely because of the small sample population. Interestingly, despite comparative analysis of 576 metabolites between these slow-growing and normal-growing littermates, the relative abundance of only 36 metabolites differed between the pairs. Most of these metabolites could be eliminated as potential markers because of the difficulty with the extraction and rapid measurement of their plasma/serum concentrations. Histamine differed from most of these potential metabolite markers in that commercial sandwich ELISAs are readily available. Using an ELISA, we verified the metabolomic data, demonstrating that plasma histamine concentrations were 150% higher in slow-growing than normal growing littermates of similar birth weight (P < 0.05). Subsequently, a separate data set was obtained using swine from a different geographical location and genetic background and also showed that elevated histamine (ng/mL) at birth is associated with increased preweaning growth rate (P = 0.009, r = 0.306, n = 9 litters). Together, the data indicate that perinatal histamine concentrations may serve as a tool to identify potentially slower growing pigs and as a serum biomarker for predicting litter growth rate.

Conjugated linoleic acid and betaine affect lipolysis in pig adipose tissue explants.

Consumers' demand of leaner meat products is a challenge. Although betaine and conjugated linoleic acid (CLA) have the potential to decrease porcine adipose tissue, their mode of action is poorly understood. The aim of the study was to determine the lipolytic effect of betaine and CLA in the adipose tissue of Iberian pigs. Adipose tissue explants from five pigs (38 kg BW) were prepared from dorsal subcutaneous adipose tissue samples and cultivated for 2 h (acute experiments) or 72 h (chronic experiments). Treatments included 100 µM linoleic acid (control), 100 µM trans-10, cis-12 CLA, 100 µM linoleic acid + 1 mM betaine and 100 µM trans-10, cis-12 CLA + 1 mM betaine (CLABET). To examine the ability of betaine or CLA to inhibit insulin's suppression of isoproterenol-stimulated lipolysis, test medium was amended with 1 µM isoproterenol ±10 nM insulin. Media glycerol was measured at the end of the incubations. Acute lipolysis (2 h) was increased by CLA and CLABET (85% to 121%; P < 0.05) under basal conditions. When lipolysis was stimulated with isoproterenol (1090%), acute exposure to betaine tended to increase (13%; P = 0.071), while CLA and CLABET increased (14% to 18%; P < 0.05) isoproterenol-stimulated lipolysis compared with control. When insulin was added to isoproterenol-stimulated explants, lipolytic rate was decreased by 50% (P < 0.001). However, supplementation of betaine to the insulin + isoproterenol-containing medium tended to increase (P = 0.07), while CLABET increased (45%; P < 0.05) lipolysis, partly counteracting insulin inhibition. When culture was extended for 72 h, CLA decreased lipolysis under basal conditions (18%; P < 0.05) with no effect of betaine and CLABET (P > 0.10). When lipolysis was stimulated by isoproterenol (125% increase in rate compared with basal), CLA and CLABET decreased glycerol release (27%; P < 0.001) compared with control (isoproterenol alone). When insulin was added to isoproterenol-stimulated explants, isoproterenol stimulation of lipolysis was completely blunted and neither betaine nor CLA altered the inhibitory effect of insulin on lipolysis. Isoproterenol, and especially isoproterenol + insulin, stimulated leptin secretion compared with basal conditions (68% and 464%, respectively; P < 0.001), with no effect of CLA or betaine (P > 0.10). CLA decreased leptin release (25%; P < 0.001) when insulin was present in the media, partially inhibiting insulin stimulation of leptin release. In conclusion, betaine and CLA produced a biphasic response regarding lipolysis so that glycerol release was increased in acute conditions, while CLA decreased glycerol release and betaine had no effect in chronic conditions. Furthermore, CLA and CLABET indirectly increased lipolysis by reducing insulin-mediated inhibition of lipolysis during acute conditions.

α-1 acid glycoprotein inhibits insulin responses by glucose oxidation, protein synthesis and protein breakdown in mouse C2C12 myotubes.

Increased plasma α-1 acid glycoprotein (AGP) is correlated with reduced growth rates in neonatal swine. The specific physiological mechanisms contributing to this relationship are unknown. This study was performed to determine if AGP can modify muscle metabolism by examining glucose oxidation and protein synthesis in the C2C12 muscle cell line. Cells were used for experiments 4 days post-fusion as myotubes. Myotubes were exposed to AGP for 24 h, with the last 4 h used to monitor 14C-glucose oxidation or to measure protein synthesis by incorporation of 3H-tyrosine. Treatment of C2C12 myotubes with mouse AGP (100 µg/ml) reduced glucose oxidation (P0.05, n=6 trials), whereas incubation with both AGP and insulin reduced 3H-tyrosine release by 15% (P<0.01, n=6 trials). First, these data indicate that the acute phase protein AGP can interact with the skeletal muscle to reduce glucose oxidation, but this is not the result of an effect on glucose transport. Second, AGP can specifically reduce protein synthesis. Lastly, AGP can inhibit insulin-stimulated glucose oxidation, protein synthesis and breakdown.

Regulation of fetuin A gene expression in the neonatal pig liver.

Fetuin A (also known as α2-Heremans-Schmid glycoprotein) is a protein primarily expressed by the liver and secreted into the blood. Previous studies have suggested that plasma concentrations of fetuin A are elevated with impaired growth rate in swine. The present study was designed to examine the relationship of porcine fetuin A with growth rate in the pig and to also elucidate the regulation of fetuin A expression by examining the hormonal and cytokine regulation of fetuin A mRNA abundance in hepatocytes prepared from suckling piglets. Quantitative real-time PCR assay was used to quantify the number of fetuin A mRNA molecules/molecule cyclophilin mRNA. Total RNA was isolated from liver of three different groups of pigs to assess changes in mRNA abundance of fetuin A: normal piglets at day 1, day 7 day 21 or 6 months of age (n=6 for each age); runt and control piglets at day 1 of age (n=4); slow growing and normal growing piglets at 21 days of age (n=8). Following birth, fetuin A gene expression increased from day 1 and 7 of age (P<0.05), and then declined at 21 days of age (P<0.05), with a much greater decline to 6 months of age (P<0.01). Fetuin A mRNA abundance was higher in runt pigs v. their normal birth weight littermates (P<0.05). Similarly, fetuin A gene expression was higher in livers of pigs that were born at a normal weight but that grew much slower than littermates with the same birth weight (P<0.05). Hepatocytes were isolated from preweaned piglets and maintained in serum-free monolayer culture for up to 72 h to permit examination of the influences of hormones, cytokines and redox modifiers on fetuin A mRNA abundance. Fetuin A gene expression was enhanced by glucagon, T3 and resveratrol (P<0.05). Growth hormone, cytokines (interleukin6, tumor necrosis factor-α) and antioxidants (N-acetylcysteine, quercertin) reduced fetuin A mRNA abundance (P<0.05). A role for fetuin A in postnatal development is suggested by the differences in fetuin A mRNA abundance between runt piglets or slow growing piglets and their normal growing sized littermates. The hepatocyte experiments suggest multiple hormones and cytokines may contribute to the regulation of fetuin A during early growth of the pig.

A sandwich ELISA for porcine alpha-1 acid glycoprotein (pAGP, ORM-1) and further demonstration of its use to evaluate growth potential in newborn pigs.

A simple, reproducible sandwich, ELISA was developed to measure porcine alpha-1 acid glycoprotein (pAGP, ORM-1) in pig plasma. Porcine AGP isolated from serum was purchased and a polyclonal antisera was prepared in rabbits using the whole pAGP molecule as immunogen. The antiserum was affinity purified, and a portion of the purified antibody fraction was labeled with horseradish peroxidase. Porcine AGP protein was used as a standard, whereas commercially available buffers and reagents were utilized throughout the assay. The assay was specific for pAGP, had a lower limit of detection of 3.2 ng/mL, and could be used to quantify pAGP in plasma or serum. Using this ELISA, we corroborated our previous findings obtained by RID assay, which demonstrated that the AGP concentration in newborn piglets is negatively associated with preweaning growth rate. The current data were obtained using piglets from a different geographical location and genetic background and showed that elevated AGP at birth was associated with reduced preweaning growth rate (P < 0.001, r = 0.433, n = 19 litters). In addition, litters with a greater average AGP at birth were at a growth disadvantage compared with litters with reduced average AGP plasma concentrations (P < 0.001, r = 0.708, n = 19 litters). Litter average plasma AGP was a better predictor of litter preweaning growth rate than average litter birth weight. The data represent further support for using perinatal AGP concentrations as a tool to identify potential slower growing pigs and as a plasma biomarker for predicting litter growth rate.

α 1-acid glycoprotein inhibits lipogenesis in neonatal swine adipose tissue.

Serum α1-acid glycoprotein (AGP) is elevated during late gestation and at birth in the pig and rapidly declines postnatally. In contrast, the pig is born with minimal lipid stores in the adipose tissue, but rapidly accumulates lipid during the first week. The present study examined if AGP can affect adipose tissue metabolism in the neonatal pig. Isolated cell cultures or tissue explants were prepared from dorsal subcutaneous adipose tissue of preweaning piglets. Porcine AGP was used at concentrations of 0, 100, 1000 and 5000 ng/ml medium in 24 h incubations. AGP reduced the messenger RNA (mRNA) abundance of the lipogenic enzymes, malic enzyme (ME), fatty acid synthase and acetyl coA carboxylase by at least 40% (P<0.001). The activity of ME and citrate lyase were also reduced by AGP (P<0.05). Glucose oxidation was reduced by treatment with 5000 ng AGP/ml medium (P<0.05). The 14C-glucose incorporation into fatty acids was reduced by ~25% by AGP treatment for 24 h with 1000 ng AGP/ml medium (P<0.05). The decrease in glucose metabolism by AGP appears to function through an inhibition in insulin-mediated glucose oxidation and incorporation into fatty acids. This was supported by the analysis of the mRNA abundance for sterol regulatory element-binding protein (SREBP), carbohydrate regulatory element-binding protein (ChREBP) and insulin receptor substrate 1 (IRS1), which all demonstrated reductions of at least 23% in response to AGP treatment (P<0.05). These data demonstrate an overall suppression of lipogenesis due to AGP inhibition of lipogenic gene expression in vitro, which the metabolic data and SREBP, ChREBP and IRS1 gene expression analysis suggest is through an inhibition in insulin-mediated events. Second, these data suggest that AGP may contribute to limiting lipogenesis within adipose tissue during the perinatal period, as AGP levels are highest for any serum protein at birth.

Regulation of alpha-1 acid glycoprotein synthesis by porcine hepatocytes in monolayer culture.

Alpha-1 acid glycoprotein (AGP, orosomucoid, ORM-1) is a highly glycosylated mammalian acute-phase protein, which is synthesized primarily in the liver and represents the major serum protein in newborn pigs. Recent data have suggested that the pig is unique in that AGP is a negative acute-phase protein in this species, and its circulating concentration appears to be associated with growth rate. The purpose of the present study was to investigate the regulation of AGP synthesis in hepatocytes prepared from suckling piglets and to provide a framework to compare its regulation with that of haptoglobin (HP), a positive acute-phase protein. Hepatocytes were isolated from preweaned piglets and maintained in serum-free monolayer culture for up to 72 h. The influences of hormones, cytokines, and redox modifiers on the expression and secretion of AGP and HP were determined by relative polymerase chain reaction and by measuring the concentration of each protein secreted into culture medium. The messenger RNA abundance and/or secretion of AGP protein was enhanced by interleukin (IL)-17a, IL-1, and resveratrol and inhibited by tumor necrosis factor-α (TNF), oncostatin M, and thyroid hormone (P < 0.05). HP expression and synthesis were upregulated by oncostatin M, IL-6, and dexamethasone and downregulated by TNF (P < 0.01). The overall messenger RNA expression at 24 h was in agreement with the secreted protein patterns confirming that control of these proteins in hepatocytes is largely transcriptional. Moreover, these data support the consideration that AGP is a negative acute-phase reactant and appears to be regulated by cytokines (with the exception of TNF) and hormones primarily in a manner opposite to that of the positive acute-phase protein, HP.

Identification and characterization of a nuclear factor-κ B-p65 proteolytic fragment in nuclei of porcine hepatocytes in monolayer culture.

Hepatic responses to proinflammatory signals are controlled by the activation of several transcription factors, including, nuclear factor-κ B (NF-κB). In this study, hepatocytes prepared from suckling pigs and maintained in serum-free monolayer culture were used to define a novel proinflammatory cytokine-specific NF-κB subunit modification. The immunoreactive p65 protein was detected by Western blot analysis at the appropriate molecular weight in the cytosol of control cultures and those incubated with tumor necrosis factor-α (TNF). However, in nuclei, the p65 antisera cross-reacted with a protein of approximately 38 kDa (termed p38) after TNF addition, which was not observed in the cytosol of control or cytokine-treated cells. Specifically, incubation with TNF also resulted in phosphorylation (P < 0.05) of the inhibitor complex protein (IκB), whereas incubation with other cytokines, IL-6, IL-17a, or oncostatin M was not associated with either phosphorylation of IκB or nuclear translocation of p65. Intracellular endothelial nitric oxide synthase was deceased (P < 0.05) and plasminogen activator inhibitor-1 secretion was increased (P < 0.05) after TNF incubation. The TNF-induced p38 protein was purified from hepatocyte nuclei by immunoprecipitation, concentrated by electrophoresis, and subsequently analyzed by mass spectrometry. Ten unique NF-κB p65 peptides were identified after digestion with trypsin and chymotrypsin; however, all were mapped to the N-terminus and within the first 310 amino acid residues of the intact p65 protein. Although low molecular weight immunoreactive p65 molecules were previously observed in various human and rodent systems, this is the first report to positively identify the p38 fragment within hepatocyte nuclei or after specific cytokine (TNF) induction.

Methyl-ß-cyclodextrin alters adipokine gene expression and glucose metabolism in swine adipose tissue.

This study was designed to determine whether methyl-ß-cyclodextrin (MCD) can substitute for albumin in incubation medium for neonatal swine adipose tissue explants, or whether MCD affects metabolism and cytokine expression. Subcutaneous adipose tissue explants (100 ± 10 mg) were prepared from 21-day-old pigs. Explants were incubated in medium 199 supplemented with 25 mM HEPES, 1.0 nM insulin at 37°C. The medium also contained bovine serum albumin (BSA) or MCD at 0%, 0.05%, 0.1%, 0.2% or 0.3%. Tissue explants were treated with these media for 1 h and then switched to the same basal incubation medium containing 0.05% BSA. Explants were removed from basal medium at 2 or 8 h of incubation, and real-time PCR was performed to assess expression of tumor necrosis α (TNF) and interleukin 6 (IL6), acetyl CoA carboxylase (ACAC) and fatty acid synthase (FASN). Alternatively, rates of 14C-glucose oxidation and lipogenesis were monitored ± insulin (100 nM), following MCD treatment. Incubation with BSA had minimal effects on gene expression or adipose tissue metabolism, only producing a doubling in TNF mRNA abundance (P < 0.01). Treatment with MCD increased TNF mRNA abundance by eightfold (P < 0.009), whereas IL6 gene expression increased a 100-fold (P < 0.001) with a suppression in ACAC and FASN expression (P < 0.01). This was paralleled by MCD inhibition of insulin-stimulated glucose oxidation and lipogenesis (P < 0.001). Addition of a TNF antibody to the incubation medium alleviated this inhibition of insulin-stimulated glucose metabolism by ~30% (P < 0.05).

Peripheral tumor necrosis factor α regulation of adipose tissue metabolism and adipokine gene expression in neonatal pigs.

The neonatal pig is susceptible to stress and infection, conditions which favor tumor necrosis factor α (TNFα) secretion. This study examined whether TNFα can alter metabolic activity and cytokine gene expression within neonatal pig adipose tissue. Cell cultures were prepared from neonatal subcutaneous adipose tissue using standard procedures. Cultures (5 experiments) were incubated with medium containing (14)C-glucose for 4 h to measure glucose conversion to lipid in the presence of combinations of TNFα (10 ng), insulin (10 nM) and an anti-pig TNFα antibody (5 µg). Basal lipogenesis was not affected by TNFα treatment (P > 0.05). However, insulin stimulated lipogenesis was reduced by TNFα (P < 0.02). For gene expression studies, cultures were incubated with 0, 2.5, 5.0 or 10 ng TNFα for 2, 4 or 24 h (n = 4 experiments). Interleukin 6 and TNFα gene expression were acutely (2-4 h) stimulated by exogenous TNFα treatment (P < 0.05), as analyzed by real-time PCR. Adiponectin mRNA abundance was reduced (P < 0.001) while monocyte chemotactic gene expression was increased by TNFα treatment at all time points (P < 0.001). Chronic treatment (24 h) was required to increase monocyte multiplication inhibitory factor or suppress lipoprotein lipase gene expression (P < 0.02). These data suggest conditions which increase serum TNFα, like sepsis, could suppress lipid accumulation within adipose tissue at a time of critical need in the neonate and induce a variety of adipose derived cytokines which may function to alter adipose physiology.

Iron dextran treatment does not induce serum protein carbonyls in the newborn pig.

Oxidation of serum proteins can lead to carbonyl formation that alters their function and is often associated with stress-related diseases. As it is recommended that all pigs reared in modern production facilities be given supplemental iron at birth to prevent anemia, and metals can catalyze the carbonylation of proteins, the primary objective of this study was to determine whether standard iron dextran treatment was associated with enhanced serum protein oxidation in newborn piglets. Piglets were treated with 100 mg of iron dextran intramuscularly either on the day of birth, or on the third day after birth. Blood samples were collected from piglets 48 or 96 h after treatment and serum was harvested. For quantification, serum protein carbonyls were converted to hydrazones with dinitrophenyl hydrazine and analyzed spectrophotometrically. To identify and determine relative distribution of carbonylated proteins, serum protein carbonyls were derivatized with biotin hydrazide, separated by two-dimensional polyacrylamide gel electrophoresis, stained with avidin-fluorescein and identified by mass spectrometry. The standard iron dextran treatment was associated with no increase in total oxidized proteins if given either on the first or third day of life. In addition, with a few noted exceptions, the overall distribution and identification of oxidized proteins were similar between control and iron dextran-treated pigs. These results indicate that while iron dextran treatment is associated with a marked increase in circulating iron, it does not appear to specifically induce the oxidation of serum proteins.

IGF-I mediated inhibition of leptin receptor expression in porcine hepatocytes.

A study was conducted to elucidate hormonal control of leptin receptor gene expression in primary cultures of porcine hepatocytes. Hepatocytes were isolated from swine and seeded into T-25 flasks. Cultures were established in medium containing fetal bovine serum for one day and switched to serum-free medium (William's E medium and 1 ng/mL insulin) for the remainder of the 3d culture period. For the final 24 h, medium was supplemented with porcine growth hormone (GH, 100 or 500 ng/mL), insulin-like growth factor 1 (IGF-1, 50 to 250 ng/mL) or triiodothyronine (T3, 100 ng/mL). RNA was extracted and relative quantitative RT-PCR was performed with primers for long form leptin receptor. Receptor expression was calculated relative to 18S rRNA. Insulin had no effect (P>0.05), while T3 increased leptin receptor mRNA abundance (P<0.05). Treatment with GH or IGF-I reduced leptin receptor expression (P<0.05). Phosphorylation of ERK1/2 in response to acute leptin treatment was inhibited by previous exposure to GH or IGF-I. Hepatocytes secreted IGF-I under basal conditions and this was enhanced by GH addition. These data suggest porcine hepatocytes may be less sensitive to leptin stimulation due to the actions of endogenous IGF-I on leptin receptor expression.

Adipokine gene transcription level in adipose tissue of runt piglets.

Runt piglets were used as a model for neonatal stress to test the hypothesis that stress during the pre-weaning period can alter adipokine gene transcription levels. Runts were selected by birth mass <1kg and compared to littermates (controls) of mean litter weight. Subcutaneous (SQ) and perirenal (PR) adipose tissues were collected at d1 (n=5), d7 (n=7) or d21 (n=9) of age. Real time PCR was used to quantify mRNA abundance for: leptin, adiponectin, interleukin 1beta (IL1beta), IL6, IL8, IL10, IL15, tumor necrosis factor alpha, haptoglobin, macrophage migration inhibitory factor (MIF), monocyte chemotactic protein, vascular endothelial growth factor and cyclophilin. Leptin and adiponectin mRNA abundance were lower, while IL1beta, IL6, IL10 and MIF mRNA abundance were higher in SQ of runts than controls at d1 (P<0.05). Leptin, IL6, IL10, haptoglobin and MIF mRNA abundance were higher in PR from runts than controls at d7 (P<0.05) and MIF mRNA abundance was elevated by 30 fold in PR of runts at d21 (P<0.001). Thus, stressors affecting neonatal runts produce different responses in adipokine gene transcription by PR and SQ than in normal sized littermates.

Ontogeny of adipokine expression in neonatal pig adipose tissue.

This study examined ontogeny of development for a range of adipokines in neonatal adipose tissue. Pigs (Sus scrofa) were selected across six litters for sampling subcutaneous (SQ) and perirenal (PR) adipose tissues at d1, d4, d7 or d21 of age and total RNA extraction. Reverse transcription and real-time PCR were used to quantify mRNA abundance for: leptin, adiponectin, interleukin 1beta (IL-1beta), IL-6, IL-8, IL-10, IL-15, tumor necrosis factor alpha (TNFalpha), haptoglobin, vascular endothelial growth factor (VEGF), macrophage migration inhibitory factor (MIF), monocyte chemoattractant protein 1 (MCP1) and cyclophilin. Leptin, adiponectin and IL-15 expression increased from d1 to d 21 of age in both SQ and PR. Haptoglobin, VEGF, MIF and IL-8 expression decreased between d1 and d4 of age in SQ. TNFalpha expression was unchanged from d1-7 and then increased at d21. IL-1beta, IL-6 and IL-10 expression were unchanged with age in SQ; whereas IL-1beta and IL-6 mRNA abundance in the PR increased with age. Analysis of the mRNA abundance for these adipokines within adipose tissue from d1 to d21 of age demonstrated that neonatal development of adipokine expression varies among the different adipokines and the internal and external sites of adipose tissue deposition (PR versus SQ).

Uncoupling protein expression in skeletal muscle and adipose tissue in response to in vivo porcine somatotropin treatment.

These experiments examined the potential roles of somatropin (pST) and IGF-I in the regulation of uncoupling protein (UCP)2 and UCP3 and their regulatory proteins peroxisome proliferator activated receptor (PPAR) alpha, gamma and delta using in vivo pST treatment of swine and in vitro supplementation of pST or IGF-I to adipose slices. Six, 90kg barrows were treated with recombinant pST (10mg) for 2 week while another six pigs were injected with buffer. Total RNA from outer subcutaneous adipose (OSQ) and middle subcutaneous adipose (MSQ) tissues, leaf fat, liver and longissimus (LM) was amplified by reverse transcription-PCR with quantification of transcripts by capillary electrophoresis with laser-induced fluorescence detection. UCP2 mRNA abundance increased in liver (P<0.001) and all three adipose tissues by pST treatment (P<0.05). Administration of pST increased UCP3 mRNA abundance by 42% in LM (P<0.01). PPARalpha mRNA abundance increased with pST treatment by 29% in liver (P<0.05), while decreasing 25% in LM (P<0.05). PPARgamma mRNA abundance decreased 32% (P<0.01) while PPARdelta increased 48% in LM (P<0.01) with pST administration. In vitro, pST reduced UCP2 mRNA abundance in OSQ and MSQ tissue slices (P<0.05). UCP3 mRNA abundance decreased in OSQ (P<0.05) but increased in MSQ (P<0.05) with pST. In contrast, IGF-I increased UCP2 and UCP3 mRNA abundance in both MSQ and OSQ slices (P<0.05). These experiments suggest pST, IGF-I and metabolic adaptations to pST contribute to regulating UCP2 and UCP3.

Impact of dietary protein content on uncoupling protein mRNA abundance in swine.

The present study was designed to determine if dietary protein can alter uncoupling protein (UCP) expression in swine, as has been shown in rats, and attempt to identify the mechanism. Eight pigs (approximately 50 kg body mass) were fed an 18% crude protein (CP) diet while another eight pigs were switched to a diet containing 12% crude protein (CP) and fed these diets until 110 kg body mass. The outer (OSQ) and middle (MSQ) subcutaneous adipose tissues, liver, leaf fat, longissimus (LM), red portion of the semitendinosus (STR) and the white portion of the ST (STW) were analyzed for gene expression by real-time PCR. Feeding of 12% CP did not alter growth or carcass composition, relative to 18% CP (P>0.05). Serum growth hormone, non-esterified fatty acids, triglycerides and urea nitrogen were reduced with the feeding of 12% CP (P<0.05). The UCP2 mRNA abundance was reduced in LM, STR, MSQ and OSQ with feeding of 12% CP (P<0.05), as was UCP3 mRNA abundance in MSQ and STW (P<0.01). Peroxisome proliferation activated receptor alpha (PPARalpha) and PPARgamma were reduced in MSQ and STR (P<0.05) with feeding 12% CP as was the PPARalpha regulated protein, acyl CoA oxidase (ACOX, P<0.05). These data suggest that feeding 12% CP relative to 18% CP reduces serum NEFA, which reduces PPARalpha and PPARgamma expression and consequently reduces UCP2 lipoperoxidation in OSQ and STR and also reduced UCP3 associated fatty acid transport in MSQ and STW.

Beta-adrenergic regulation of uncoupling protein expression in swine.

This study examined the beta-adrenergic regulation of uncoupling protein (UCP) 2 and UCP3 gene expression in porcine tissues. In vitro experiments examined changes in UCP2 and UCP3 gene expression in middle (MSQ) and outer (OSQ) subcutaneous adipose tissues from crossbred neutered male pigs. Incubation of tissue slices (24 h) with 0 to 1000 nM isoproterenol increased UCP2 and UCP3 mRNA abundance in MSQ and OSQ, relative to 18S rRNA (P<0.05). For the in vivo experiment, nine randomly selected pigs (80 kg) were presented with a diet supplemented with 10.0 ppm ractopamine for 2 weeks. Another eight pigs were maintained on a control diet. Dietary ractopamine did not affect adipose UCP2 or UCP3 gene expression (P>0.05). However, UCP2 mRNA abundance was depressed in semitendinosus white (STW, P<0.05) and semitendinosus red (STR, P<0.001) by ractopamine feeding. Also, ractopamine decreased UCP3 mRNA abundance by 28% in STW (P<0.05). The in vitro data suggest that beta-adrenergic agonists directly affect adipose tissue UCP expression, although these adipose effects can be masked by the in vivo physiology. The in vivo data indicate that beta-adrenergic agonists may function in regulating UCP2 and UCP3 expression in selected muscles.

Leptin and leptin receptor expression in skeletal muscle and adipose tissue in response to in vivo porcine somatotropin treatment.

The present study was performed to examine the response of leptin and leptin receptor (Rb) genes to porcine somatotropin (pST) stimuli in finishing pigs. Twelve crossbred barrows (Yorkshire x Landrace) were used in this study. Animals were individually fed a basal diet containing 18% CP, 1.2% lysine, and 3.5 Mcal of DE/kg ad libitum (as-fed basis). At 90 kg, six pigs were treated with daily injections of recombinant pST (10 mg) in sterile bicarbonate buffer, whereas the other six pigs were injected with sterile bicarbonate buffer (controls). With initiation of pST treatment, the quantity of feed offered was 85% of calculated ad libitum intake based on BW and adjusted every 3 d. Diet restriction was designed to correct for the effects of the known inhibition in feed intake because of pST treatment in swine. Animals were maintained on treatment for 2 wk. A blood sample was obtained from each pig on d 14 of treatment, 6 h after pST injection. Tissue samples were collected on d 15, frozen in liquid N2, and stored at -80 degrees C before analysis for mRNA abundance. Total RNA was amplified by reverse transcription (RT) PCR with subsequent quantification of transcripts by capillary electrophoresis with laser-induced fluorescence detection. Samples included outer subcutaneous adipose tissue (OSQ), middle subcutaneous adipose tissue (MSQ), leaf fat (LF), liver, latissimus dorsi (LD), and biceps femoris (BF). Restricted feeding resulted in no change in BW of control pigs, whereas pST treatment increased BW by 6.9 +/- 0.5 kg (P < 0.001). Treatment with pST produced a 12-fold increase in serum ST concentration relative to control pigs (P < 0.002). Serum leptin concentration was increased by 17% in swine treated with pST relative to control pigs (P < 0.011). Leptin mRNA abundance was increased in liver by pST treatment (P < 0.05). Administration of pST decreased leptin Rb (Ob-Rb) mRNA abundance by 27% in liver (P < 0.044) and by 49.5% in OSQ (P < 0.025) relative to controls. The present data suggest that pST does not affect leptin expression independent of dietary intake because the restricted feeding regimen used in the present study precluded detection of major change in leptin gene expression. Changes in Ob-Rb mRNA abundance by pST treatment indicate that ST or the metabolic adaptations to ST have a role in regulating Ob-Rb expression.

Porcine preadipocyte proliferation and differentiation: a role for leptin?

The present study was designed to determine whether porcine leptin can alter the proliferation and differentiation of the porcine preadipocyte. The stromal vascular cell fraction of neonatal pig s.c. adipose tissue was isolated by collagenase digestion, filtration, and subsequent centrifugation. For differentiation studies, cells were seeded on six-well tissue culture plates and proliferated to confluency in 10% (vol/vol) fetal bovine serum (FBS) in Dulbecco's modified Eagle medium/F12 (DMEM/F12; 50:50). Cultures were differentiated using 2.5% pig serum (vol/vol) and recombinant porcine leptin at concentrations of 0 to 1,000 ng/mL alone or in combination with porcine insulin (100 nM), dexamethasone (1 microM), or IGF-1 (250 ng/mL). After 7 d of lipid filling, cultures were harvested for analysis of sn-glycerol 3 phosphate dehydrogenase (GPDH) and lipoprotein lipase (LPL). The GPDH and LPL activities are measures of preadipocyte differentiation. Data were corrected for protein content of the cultures. For proliferation experiments, 24 h after seeding cells with 10% FBS in DMEM/F12 in 25-cm2 tissue culture flasks, cells were switched to 5% FBS and supplemented with 0 to 1,000 ng of porcine leptin or 1,000 ng of murine leptin. Cell proliferation was measured by 3H-thymidine incorporation in preconfluent cultures over 24 h on d 4 of culture. At confluency, cells were switched to a medium to promote differentiation and lipid filling (2.5% pig serum, 100 nM insulin, 1 microM dexamethasone) for 7 d. Cells were harvested from the flasks and adipocytes were separated from stromal cells by Percoll gradient centrifugation. In a series of experiments, leptin alone or in combination with insulin, dexamethasone, or IGF-I did not affect differentiation as measured by the activity of GPDH and LPL. Leptin at any concentration did not inhibit differentiation induced by insulin, dexamethasone, or IGF-I; however, leptin at 1,000 ng/mL stimulated a 30% increase in preadipocyte proliferation (P = 0.007; n = 6) and a 27% increase in stromal cell proliferation (P < 0.001; n = 6). These results indicate that, at most, porcine leptin may contribute to the recruitment of new adipocytes within the adipose tissue.