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The importance of the microbiota in the gastrointestinal tract of animals is recognized as a critical player in host health. Recently, the significance of the mycobiome has been recognized, but culture-independent studies are limited, especially in swine. Weaning is a time of stress, dietary changes, and a predisposition to infections, making it a time point of interest to industry. In this pilot study, we sought to assess and characterize the mycobiome in the feces of swine from birth through the critical weaning transition to investigate the mycobiome population and its temporal dynamics in piglet feces. Cultured fecal samples demonstrate a significant increase in fungal burden following weaning that does not differ from adult levels, suggesting stable colonization. Culturable fungi were not found in any environmental samples tested, including water, food, sow milk or colostrum. To determine the fungal diversity present and to address the problem of unculturable fungi, we performed a pilot study utilizing ITS and 16S rRNA focused primers for high-throughput sequencing of fungal and bacterial species, respectively. Bacterial populations increase in diversity over the experimental timeline (days 1 to 35 postbirth), but the fungal populations do not demonstrate the same temporal trend. Following weaning, there is a dynamic shift in the feces to a Saccharomycetaceae-dominated population. The shift in fungal population was because of the dominance of Kazachstania slooffiae, a poorly characterized colonizer of animal gastrointestinal tracts. This study provides insights into the early colonization and subsequent establishment of fungi during the weaning transition in piglets. Future studies will investigate the effect of the mycobiome on piglet growth and health during the weaning transition.

Staphylococcus aureus is an opportunistic pathogen historically considered a minor contributor to porcine illness, but recent work has pointed to the potential of pigs as a source of zoonotic methicillin-resistant S. aureus (MRSA) infections. S. aureus is adept at acquiring antibiotic-resistance genes and has become a pathogen of interest due to its implications in pig and human health. The innate immune response plays a critical role in controlling gram positive bacterial infections (such as S. aureus) in the intestine. To elucidate the acute immune response to gram positive infections, we challenged a porcine intestinal cell line (IPEC-J2) with gram-positive antigens isolated from S. aureus. IPEC-J2 cells were grown in collagen-coated transwell dishes until a transepithelial electrical resistance (TEER) reading of >1 kΩcm(2) was obtained. Cells were then challenged with 100ug/ul peptidoglycan (PGN) or 10ug/ul lipoteichoic acid (LTA) for 3, 8 or 24 hours prior to cell and supernatant collection. Butyrate is a short chain fatty acid in the gut that has known protective effects on barrier function and intestinal health. To test the protective effect of butyrate on gram-positive intestinal infections, IPEC-J2 cells were pre-treated with butyrate for 24 hours prior to PGN or LTA challenge. Pre-treatment with butyrate prior to PGN or LTA challenge resulted in statistically significant increases of IL-1RA. After antigen challenge alone, levels of IL-1RA and IL-8 did not increase significantly over time in the cellular supernatant. IL-1b, IL-4, IL-2, IL-6, IL-10, IL-12, and IL-18 were not significantly altered by antigen challenge +/- butyrate pre-treatment. These data indicate an acute inflammatory response is produced by IPEC-J2 cells upon exposure to antigens from gram-positive bacteria, but pre-treatment with butyrate promotes an anti-inflammatory response, that may reduce intestinal damage, through increased IL-1RA secretion.

This study was designed to determine if normal birth weight pigs that grow poorly during the pre-weaning period have altered plasma metabolites that might serve as markers for prediction of growth rate. Eight pairs of average birth weight pigs (1.57 ± 0.05 kg) were identified that diverged in weight by a minimum of 50 g/day until 21 days of age. At 21 days, slow growing (SG) pigs weighed 5.47 ± 0.22 kg while control (C) littermates weighed 6.98 ± 0.28 kg (P < 0.001). Plasma was collected for analysis at days 1 and 21 postpartum for metabolomic analysis by ultrahigh performance liquid chromatography-tandem mass spectroscopy (Metabolon; Durham, NC). Analysis of the plasma from these SG pigs identified 578 metabolites, but only 36 were affected by potential growth rate at day 1 of age (P < 0.05). Plasma analysis at 21 days of age revealed that the concentration of only 28 metabolites were affected by growth rate (P < 0.05). The few plasma metabolite changes did not indicate changes in major metabolic pathways (P > 0.05). Principle component analysis revealed that groups clustered strongly by age, with all day 1 samples strongly segregating along component axis 1 from day 21 samples. Conversely, growth rate had very little effect in further segregating samples, as plasma samples from slow- versus fast-growing pigs forming largely overlapping populations. The results indicate that while age profoundly affects global metabolic profiles, growth rate elicits minimal alterations in global biochemical makeup. Examination of the data for individual plasma metabolites at birth by analysis using 2-way ANOVA with repeated measures identified elevated erythronate (P = 0.009), histamine (P = 0.019), 7-ketolithocholate (P = 0.020), serotonin (P = 0.0257); and s-adenosylhomocysteine (P = 0.001) as potential markers to identify underperforming piglets and their littermates at birth.

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.

This study evaluated the potential mechanism(s) by which leptin treatment inhibits loss of muscle mass with fasting. Cultures of C2C12 myoblasts were differentiated into myotubes with 5% (vol/vol) horse serum in Dulbecco's modified Eagle's medium/F12. These myotubes were used to assess 3H-tyrosine incorporation and release following incubation with recombinant porcine leptin (0 to 500 ng/mL). Protein synthesis in myotubes, as measured by 3H-tyrosine incorporation, was not affected by leptin treatment (P > 0.05). Protein breakdown in C2C12 myotubes, as measured by 3H-tyrosine release, was inhibited by leptin treatment. A leptin concentration of 0.5 ng/mL was sufficient to inhibit 3H-tyrosine release by 3.5% (P 0.05) on palmitate metabolism. Chronic leptin exposure resulted in up to a 26% increase in palmitate oxidation (P < 0.05). The stimulation of fatty acid oxidation with chronic leptin treatment suggests that leptin spares other energy sources in muscle from oxidation during periods of a leptin-induced decrease in feed intake.

The present study examined whether recombinant porcine leptin alters lipid synthesis in porcine adipocytes. The stromal-vascular cell fraction of neonatal pig subcutaneous adipose tissue was isolated by collagenase digestion, filtration, and subsequent centrifugation. These cells were seeded on 25-cm 2 tissue culture flasks and proliferated to confluency in 10% (vol/vol) fetal bovine serum in Dulbecco's modified Eagle medium/F12 (DMEM/F12, 50:50). Cultures were differentiated using 2.5% pig serum (vol/vol), 10 nM insulin, 100 nM hydrocortisone. After 7 d of lipid filling, cultures were washed free of this medium, incubated overnight in DMEM/F12 containing 2% pig serum (vol/vol), and then used for experiments. Acute experiments assessed U- 14 C-glucose or 1- 14 C-palmitate metabolism in cultures exposed to porcine leptin (0 to 1,000 ng/mL medium) for 4 h. Chronic experiments used cultures incubated with 0 to 1,000 ng porcine leptin/mL medium for 44 h before measurements of U- 14 C-glucose and 1- 14 C-palmitate oxidation and incorporation into lipid. Another experiment examined whether chronic leptin treatment alters insulin responsiveness by including insulin (10 nM) with incubations containing leptin. Leptin had no acute effects on glucose oxidation or conversion to lipid (P > 0.05). Acute leptin treatment decreased palmitate incorporation into lipids up to 45% (P 0.05) by leptin. These data indicate that leptin functions to promote partitioning of energy away from lipid accretion within porcine adipose tissue by inhibiting glucose oxidation and lipogenesis indirectly, by decreasing insulin-mediated stimulation of lipogenesis, and by stimulating fatty acid oxidation while inhibiting fatty acid esterification.

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 (P0.001). Treatment with pST produced a 12-fold increase in serum ST concentration relative to control pigs (P0.002). Serum leptin concentration was increased by 17% in swine treated with pST relative to control pigs (P0.011). Leptin mRNA abundance was increased in liver by pST treatment (P0.05). Administration of pST decreased leptin Rb (Ob-Rb) mRNA abundance by 27% in liver (P0.044) and by 49.5% in OSQ (P0.025) 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.

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 (P0.001; n = 6). These results indicate that, at most, porcine leptin may contribute to the recruitment of new adipocytes within the adipose tissue.

The current study was performed to examine the response of the leptin gene to hormonal stimuli in porcine adipose tissue from finishing pigs. Yorkshire gilts (approximately 150 kg BW) were used in this study. Tissue from four to six pigs was used per experiment. Dorsal subcutaneous adipose tissue samples were acquired, and adipose tissue explants (approximately 100 mg) were prepared using sterile technique. Tissue slices were transferred to 12-well tissue culture plates containing 1 mL of Media 199 with 25 mM HEPES, 0.5% BSA, pH 7.4, and various hormone supplements. Triplicate tissue slices were incubated with either basal medium or hormone-supplemented media in a tissue culture incubator at 37 degrees C with 95% air:5% CO2. Hormones included insulin (100 nM), dexamethasone (1 microM), porcine GH, 100 ng/mL), triiodothyronine (T3, 10 nM), porcine leptin (100 ng/mL), or IGF-I (250 ng/mL). Following incubation for 24 h, tissue samples from the incubations were blotted and transferred to microfuge tubes, frozen in liquid N, and stored at -80 degrees C before analysis for gene mRNA abundance by reverse-transcription PCR and subsequent quantification of transcripts by capillary electrophoresis with laser-induced fluorescence detection. Media from the incubations were collected in microfuge vials and stored at -20 degrees C before analysis for leptin content by RIA. Insulin was required to maintain tissue and mRNA integrity; therefore, insulin was included in all incubations. The combination of insulin and dexamethasone stimulated leptin secretion into the medium by 60% (P0.05; n = 6). Porcine GH inhibited insulin induced leptin secretion by 25% (P0.05; n = 6). Dexamethasone in combination with insulin produced a 22% increase in leptin mRNA abundance relative to insulin (P0.05; n = 4), and T3 stimulated a 28% increase in insulin-induced leptin mRNA abundance (P0.05; n = 4). Leptin receptor mRNA abundance was decreased by 25% with the combination of insulin and dexamethasone, relative to insulin-treated adipose tissue slices (P0.05; n = 4). Porcine GH decreased leptin receptor mRNA abundance by 17% (P0.05; n = 6). These data suggest that leptin secretion is a regulated phenomenon and that posttranslational processing may be significant. Alternatively, transport and exocytosis of leptin containing vesicles in the pig adipocyte may be quite complicated, which could account for the differences in observed mRNA abundance and protein secretion.

This study evaluated the potential mechanism(s) by which leptin treatment inhibits loss of muscle mass with fasting. Cultures of C2C12 myoblasts were differentiated into myotubes with 5% (vol/vol) horse serum in Dulbecco's modified Eagle's medium/F12. These myotubes were used to assess 3H-tyrosine incorporation and release following incubation with recombinant porcine leptin (0 to 500 ng/mL). Protein synthesis in myotubes, as measured by 3H-tyrosine incorporation, was not affected by leptin treatment (P0.05). Protein breakdown in C2C12 myotubes, as measured by 3H-tyrosine release, was inhibited by leptin treatment. A leptin concentration of 0.5 ng/mL was sufficient to inhibit 3H-tyrosine release by 3.5% (P0.05); 50 ng/mL produced a maximal inhibition of 10.2% (P0.05). Dexamethasone (1 microM) was used to maximally stimulate protein breakdown. Leptin (50 ng/mL leptin) decreased dexamethasone-induced 3H-tyrosine release by 32% (P0.05). The inhibition of 3H-tyrosine release in C2C12 myotubes suggests that leptin produces a protein-sparing effect in vitro by inhibiting protein breakdown. Fatty acid metabolism also was investigated because fatty acids are a major energy source for muscle during periods of reduced intake, as occurs with leptin treatment. Acute (4 h) and chronic (24 h) exposures to porcine leptin (0 to 500 ng/mL) were used to evaluate 14C-palmitate oxidation. Acute leptin treatment had no effect (P0.05) on palmitate metabolism. Chronic leptin exposure resulted in up to a 26% increase in palmitate oxidation (P0.05). The stimulation of fatty acid oxidation with chronic leptin treatment suggests that leptin spares other energy sources in muscle from oxidation during periods of a leptin-induced decrease in feed intake.

The present study examined whether recombinant porcine leptin alters lipid synthesis in porcine adipocytes. The stromal-vascular cell fraction of neonatal pig subcutaneous adipose tissue was isolated by collagenase digestion, filtration, and subsequent centrifugation. These cells were seeded on 25-cm2 tissue culture flasks and proliferated to confluency in 10% (vol/vol) fetal bovine serum in Dulbecco's modified Eagle medium/F12 (DMEM/F12, 50:50). Cultures were differentiated using 2.5% pig serum (vol/vol), 10 nM insulin, 100 nM hydrocortisone. After 7 d of lipid filling, cultures were washed free of this medium, incubated overnight in DMEM/F12 containing 2% pig serum (vol/vol), and then used for experiments. Acute experiments assessed U-(14)C-glucose or 1-(14)C-palmitate metabolism in cultures exposed to porcine leptin (0 to 1,000 ng/mL medium) for 4 h. Chronic experiments used cultures incubated with 0 to 1,000 ng porcine leptin/mL medium for 44 h before measurements of U-(14)C-glucose and 1-(14)C-palmitate oxidation and incorporation into lipid. Another experiment examined whether chronic leptin treatment alters insulin responsiveness by including insulin (10 nM) with incubations containing leptin. Leptin had no acute effects on glucose oxidation or conversion to lipid (P0.05). Acute leptin treatment decreased palmitate incorporation into lipids up to 45% (P0.05). Chronic leptin exposure decreased glucose oxidation (21%), total lipid synthesis (18%), and fatty acid synthesis (23%) at 100 ng/mL medium (P0.05). Insulin increased rates of glucose oxidation, total lipid, and fatty acid synthesis (P0.05); however, chronic exposure to 10 ng leptin/mL medium decreased the effectiveness of 10 nM insulin to affect these measures of glucose metabolism by approximately 18 to 46% (P0.05). Higher concentrations of leptin inhibited all effects of insulin on glucose metabolism (P0.05). Chronic exposure to leptin increased palmitate oxidation by 36% (P0.05). Chronic leptin exposure decreased palmitate incorporation into total lipids by 40% at 100 ng/mL medium (P0.05). Lipoprotein lipase activity was not affected (P0.05) by leptin. These data indicate that leptin functions to promote partitioning of energy away from lipid accretion within porcine adipose tissue by inhibiting glucose oxidation and lipogenesis indirectly, by decreasing insulin-mediated stimulation of lipogenesis, and by stimulating fatty acid oxidation while inhibiting fatty acid esterification.

The present study determined whether porcine leptin can alter the lipolytic rate in porcine adipocytes produced in vitro. The stromal-vascular cell fraction of neonatal subcutaneous adipose tissue was isolated by collagenase digestion, filtration, and subsequent centrifugation. These stromal-vascular cells were seeded on 25-cm2 tissue culture flasks and proliferated to confluency in 10% fetal bovine serum in DMEM/F12 (50:50). Cultures were differentiated using 2% pig serum + 10 mM isobutyl methylxanthine + 1 microM dexamethasone for 48 h. This medium was replaced with 5% pig serum + 1 microM insulin to promote lipid filling of adipocytes for 7 d. Adipocyte-containing cultures were incubated overnight in serum-free medium and then used for experiments. Acute experiments assessed lipolysis in cultures exposed to porcine leptin (0 to 1,000 ng/mL medium) for 2 h. Chronic experiments used cultures incubated with 100 ng porcine leptin/mL of medium for 72 h prior to lipolysis measurements. Direct effects of leptin were examined by incubating cultures in DMEM/F12, 25 mM HEPES, 3% bovine serum albumin, 20 mU of adenosine deaminase/mL of medium in the presence of 0 to 1,000 ng of porcine leptin/mL of medium. Indirect effects of leptin were examined using the same incubation medium but also supplemented with 1 microM isoproterenol +/- 10 nM insulin in the presence of 0 to 1,000 ng of porcine leptin/mL of medium. Media glycerol concentration was measured at the end of 2-h incubations. Acute leptin exposure induced up to a 76% increase in lipolysis (P < 0.05) but had no effect on insulin's inhibition of lipolysis. Chronic exposure to leptin produced up to a 56% increase in lipolysis (P < 0.05) and reduced insulin's inhibition ofisoproterenol-stimulated lipolysis by up to 31% (P < 0.05). These data demonstrate leptin functions to promote the partitioning of energy away from lipid accretion within porcine adipose tissue by promoting lipolysis directly and indirectly by reducing insulin-mediated inhibition of lipolysis.

Diabetes induced during gestation has previously been demonstrated to increase adipose accretion in the fetal pig. The present experiment examined whether maternal diabetes alters the proliferation and differentiation of the fetal preadipocyte. Seven crossbred gilts were injected with alloxan (50 mg/kg) at d 75 of gestation to induce diabetes and seven additional gilts were injected with buffer (controls). All gilts underwent Caesarean section of d 105 of gestation. Cells obtained from adipose tissue of fetuses of diabetic swine (FDS) at d 105 of gestation demonstrated a greater (P < .05) proliferative response (57%) and higher (P < .05) rates of differentiation as determined by sn-glycerol-3-phosphate dehydrogenase (142% increase) and lipoprotein lipase (80% increase) activities than cells acquired from fetuses of control swine (FCS). Adipogenic activity of the sera from these two groups of fetuses did not differ when tested on adipose tissue from fetuses at 105 d of gestation. However, use of these sera on cells derived from normal fetuses at 75 d of gestation resulted in detection of an increase (P < .05) in adipogenic activity within the sera from FDS. This study suggests that maternal diabetes causes alterations in the preadipocyte fraction of cells within the developing adipose tissue that result in formation of more adipocytes and thus permits greater capacity for lipid accumulation in the growing fetus of the diabetic pig. These alterations in the preadipocyte result from the activity of factors that transitionally function during the latter half of gestation.

Two experiments were performed to determine whether esterification is a major pathway of fatty acid utilization within porcine placenta and to determine what metabolic parameters may limit fatty acid transfer to the fetal pig. Maternal (endometrium) and fetal (chorioallantois) placenta were obtained by Caesarean section at d 110 of gestation in both experiments. Eight gilts were used in the first experiment. Tissue sections were incubated with palmitate at concentrations ranging from .25 to 2.0 mM. Maternal placenta metabolized palmitate at a higher rate than fetal placenta, although fetal placenta was more efficient in esterifying palmitate. Esterification composed the majority of palmitate utilization within fetal and maternal placenta. The second experiment evaluated the effect of dietary lipid on placental fatty acid metabolism and evaluated the ability of placenta to mobilize lipids. Fourteen gilts were divided into two groups of seven and fed a diet containing 15% tallow diet or a diet not supplemented with tallow (control) from d 90 to 110 of gestation. Dietary lipid had no detectable effects on lipoprotein lipase activity, [14C]palmitate metabolism, or lipolysis by the maternal or fetal placenta. Lipolytic activity of placental tissues was minimally affected by incubation with various proposed lipolytic activity of placental tissues was minimally affected by incubation with various proposed lipolytic agents. The data indicate that supply of fatty acids to the fetal pig may be limited by transfer of plasma fatty acids into the cytoplasm of placental cells or by regulatory enzymes for intermediate esterification; both types of limitations have been proposed to be influenced by fatty-acid binding proteins.

This experiment was designed to determine whether mobilizing maternal energy stores by fasting pregnant gilts would promote fetal energy storage by altering placental and fetal lipid metabolism. Pregnant gilts were fed a 15% tallow diet from d 80 to 99 and then fed a basal high-carbohydrate diet (control) or fasted from d 100 to 110 of gestation. Caesarean section was performed on d 110. Fasting caused maternal nonesterified fatty acid (NEFA) levels to increase 7.5-fold, beta-hydroxybutyric acid (beta-HBA) levels to increase 4.8-fold triglyceride (TG) levels to decrease 1.8-fold, and no change in plasma glucose concentration compared with controls. Fasted fetuses had a 1.3-fold increase in NEFA, 1.9-fold decrease in TG, 1.5-fold decrease in glucose, and no change in beta-HBA levels compared with control fetuses. Distribution of NEFA in fetal plasma was different from distribution of NEFA in maternal plasma. Esterification of [14C]-palmitate by maternal placenta and fetal adipose tissue was reduced by fasting, but other parameters of fatty acid metabolism were unaffected. Fasting decreased lipoprotein lipase activity per milligram of protein by 33% in maternal placenta and by 44% in fetal adipose tissue. Glycogen content of fetal liver and skeletal muscle was reduced by fasting pregnant gilts, but there was no detectable effect on percentage of carcass lipid of the fetus. These data suggest that fasting mobilizes maternal fuel stores but that these stores are not effectively used by the placenta or transported to the fetus for storage.

We investigated the effects of weaning or fasting of 21- or 35-d-old swine by monitoring serum mitogenic activity, circulating insulin-like growth factor I (IGF-I) and its binding proteins using L6 myoblast bioassays, RIA and ligand blotting techniques. Serum samples were collected from 21- or 35-d-old animals just before and 36 h after weaning or fasting. Sera from 21- and 35-d-old weaned animals were not significantly altered in their ability to promote myoblast proliferation, whereas sera from 21- and 35-d-old fasted animals caused 29 and 21% decreases (P less than .05) compared with preweaning. The mitogenic activity of control serum was inhibited by serum from fasted animals but not by preweaned or weaned sera. Serum IGF-I levels were decreased 65 to 70% (P less than .05) with weaning or fasting at both ages. Unoccupied binding sites on circulating IGF binding proteins in the 155 kDa range decreased 18 to 19% with weaning at both ages and decreased 40% (P less than .05) with fasting at 21 d but only 17% at 35 d. Ligand blotting revealed that the 43 and 39 kDa IGF binding protein bands decreased with weaning and fasting at both ages, whereas the 29-kDa band increased with weaning and fasting. These data indicate that serum IGF-I and specific IGF binding protein bands decrease during weaning or fasting at 21 and 35 d of age. However, serum mitogenic activity did not always follow serum IGF-I levels.

The dietary dose responsiveness of conjugated linoleic acid (CLA) addition relative to the fatty acid profile of edible lean tissue was examined in grower pigs treated with or without porcine somatotropin (pST). Gilts and barrows were fed CLA at 0, 0.25, 0.5, 1.0, or 2.0% of diet by weight from 20 to 55 kg BW. Additional pigs were administered (pST) at 0 or 100 microg x kg BW x d(-1) and fed either 0.5 or 2.0% CLA. Animals were fed diets containing 18% CP, 1.2% lysine, and 3.5 Mcal of DE/kg at 110% of ad libitum intake. The fatty acid profile in latissimus dorsi and dorsal s.c. adipose tissue samples was determined by gas chromatography. Dietary CLA replacement of corn oil increased the percentage of total fatty acids as stearic acid, whereas the percentages as oleic and linolenic acids were reduced in lattisimus muscle. Treatment with CLA + pST increased the percentages of linoleic and arachidonic acids while reducing the percentages of palmitic and oleic acids in lattisimus muscle. Dietary CLA increased the percentages of palmitic and stearic acids in s.c. adipose tissue while reducing the percentages of oleic, linoleic, linolenic, and arachidonic acids. The percentage of palmitic acid was reduced in s.c. adipose tissue, whereas linoleic acid was increased with CLA + pST. No synergistic effect was detected between CLA and pST for reducing carcass lipid content in grower pigs. However, pST increased the percentage of polyunsaturated fatty acids in lattisimus muscle and s.c. adipose tissue while reducing the percentages of saturated fatty acids in swine fed CLA.

The purpose of these studies was to determine the time course for changes in feed intake, blood metabolites, and lipogenic activity in adipose tissue in response to the initiation of porcine somatotropin (pST) treatment and following withdrawal from treatment in barrows. An initial study was conducted to determine the impact of chronic pST treatment (4 wk of daily injection; 0 vs 4 mg/d) on adipose tissue lipid metabolism in barrows (initial weight 67 kg). Feed efficiency was improved 27%, backfat thickness was decreased 43%, and glucose and lactate oxidation and incorporation into lipid in adipose tissue was reduced 70 to 86% in pST-treated pigs. Palmitate esterification was decreased 44%, whereas palmitate oxidation was unaffected. In vitro metabolism of lactate, glucose, and palmitate in liver slices was not affected by pST treatment. The time-course for changes in intake and adipose tissue metabolism in response to 7 d of pST (0 vs 4 mg/d) treatment and 7 d of withdrawal was examined in subsequent studies in barrows (initial weight 75 kg). Feed intake during pST treatment was significantly (P.05) less than in control pigs within 24 h of the initiation of treatment and remained low through 3 d after withdrawal. Adipose tissue biopsies were obtained on d 0, 1, 2, 4, and 7 of the treatment phase and on d 2, 4, and 7 after withdrawal from 7 d of treatment. Maximal inhibition of lipogenesis by pST treatment in adipose tissue in vitro was observed on d 4 (-68%) and d 7 (-69%). Similarly, fatty acid synthase activity declined during the treatment period, with the greatest change noted on d 7 (-26%). After withdrawal from treatment, lipogenesis gradually increased, returning to control values 7 d after withdrawal. Levels of IGF-I began to increase from d 1 to d 7 of treatment, continually decreased during withdrawal, and were normalized by the end of the withdrawal period. Plasma urea nitrogen concentrations decreased during treatment, increased during the withdrawal phase, and were normalized 4 d after the last pST treatment. Overall results indicate that most of the metabolic changes in response to pST occur within 1 wk of treatment and return to pretreatment values after 7 d of withdrawal from treatment.

The relationship between leptin mRNA and insulin status was explored using streptozotocin diabetic pigs. Twelve male Yorkshire x Landrace crossbred swine (approximately 40 kg BW) were divided into three groups. Two groups were rendered diabetic with the use of streptozotocin (75 mg/kg BW). Diabetes was confirmed 24 h after streptozotocin treatment by the presence of hyperglycemia. One group of diabetic animals received daily injections of insulin (.5 U/(kg x d)(-1)) for 7 d, whereas the other group of diabetic animals received saline injections. The nondiabetic group also received saline injections (controls). Tissue and blood were collected after 7 d of treatment. Leptin mRNA concentrations in dorsal s.c. adipose tissue were measured by Northern analysis and standardized against 28S rRNA expression. Diabetes reduced leptin mRNA concentration by 67% in s.c. adipose tissue (P < .05). Serum insulin concentrations in the diabetic animals were reduced by 69% (P < .05). Insulin treatment of diabetic animals resulted in an increase in leptin mRNA concentration to levels in controls. Primary cell culture of porcine adipose tissue was used to assess whether these actions were the direct or indirect action of insulin. Acute exposure (1 to 24 h) of primary cultures of porcine adipocytes to insulin did not result in a change in leptin expression. However, chronic (7-d) exposure to insulin elevated leptin mRNA levels by 73%. These data suggest that insulin mediates changes in porcine leptin mRNA levels in vivo or in vitro, most likely by an indirect action.

Leptin is a 16-kDa protein secreted by adipocytes that has been proposed to regulate feed intake in mice, rats, and humans. The present study was designed to characterize porcine leptin structure and expression. Successful RT-PCR resulted in development of a cDNA clone to the full length coding region of porcine leptin. Sequence data demonstrate 85% base homology to rodent, 88% to human, and a 92% homology to the bovine sequence. For assessment of porcine leptin gene expression, total RNA was extracted from the subcutaneous adipose tissue of genetically selected high backfat pigs and from contemporary crossbred swine. Total RNA derived from genetically selected high fat pigs contained 113% higher (P < .05) concentrations of porcine leptin mRNA than total RNA derived from contemporary crossbred pigs. Western blotting was used to evaluate serum levels of porcine leptin in genetically selected high backfat and contemporary, crossbred pigs. Relative levels of porcine leptin in sera from obese swine were approximately 306% higher (P < .05) than levels present in sera from contemporary, crossbred swine. These data indicate that leptin is expressed in pigs, the expressed protein is secreted into the bloodstream, and obese swine express higher levels of leptin mRNA and protein than nonobese swine at similar body weight.

The development of adipocytes was studied in primary cultures of rat adipose tissue stromal-vascular cells on collagen-coated glass coverslips. The effects of cell density and serum source on lipoprotein lipase (LPL), esterase and lipid histochemistry were evaluated. With a mixture of fetal calf serum (FCS; 2%), horse serum (2%) and pig serum (PS; 10%), large and loosely arranged clusters of adipocytes developed with time through an increase in cell number and size. An inverse relationship was observed between cell size and the number of adipocytes in a cluster. Lower cell densities were associated with large cells and the densest areas contained smaller cells. Unilocular adipocytes were observed by d 13 after plating and were generally absent from the densest area of the coverslips. Histochemically detectable LPL activity was demonstrable before lipid deposition in adipocyte clusters. A comparison of FCS (10%) and PS (10%) as the only serum sources indicated higher level of adipocyte esterase activity and lipid deposition in PS cultures. Cultures of cells from weanling (21 to 28 d old) and old (18 mo) rats were similar, whereas cells from younger rats (2 to 4 d old) formed denser cultures that contained fewer adipocytes. These adipocytes were small (less than 30 micron) and morphologically homogeneous (all multilocular). When cells from the very young rats (2 to 4 d old) were plated at low densities, an inverse relationship between cell size and number of cells in a cluster was also observed.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of insulin-like growth factor I (IGF-1) in the development of the porcine preadipocyte was studied. Primary cultures of stromal-vascular cells (containing preadipocytes) were derived from s.c. adipose tissue of pigs at 1 d of age by enzyme digestion and centrifugation. Cells were cultured for a total of 15 d. Cells were exposed to IGF-1 at concentrations of 0, 5, 25 or 50 ng/ml medium during one of four time periods: d 1-15, d 1-5, d 13-15, or 4 h on d 15 of culture. IGF-1 had a mitogenic effect on cells during the first three time periods as determined by coulter counting. IGF-1 induced the enzymatic differentiation of porcine preadipocytes following exposure for either the entire 15 d of culture or for only 48 h (d 13-15) after confluency had been attained (d 5). Histochemically, lipid accumulation over time paralleled changes in enzyme activity. Incubation of IGF-1 with the cell cultures during the logarithmic phase of growth (d 1-5) or for 4 h on d 15 did not affect enzyme activity. These data indicate that IGF-1 can induce the differentiation of porcine preadipocytes after the cells leave the logarithmic phase of growth through action on post-confluent events.

Experiments were performed to determine whether central endocrine or neural regulation is primarily involved with the development of endocrine responses in fetal adipose tissue metabolism. Fetuses within one uterine horn were either decapitated (decap) or spinally cauterized at 45 d of gestation, with fetuses in the other horn serving as sham controls (intact). Fetuses were removed by cesarean section at 110 d of gestation. Slices of subcutaneous adipose tissue (100 mg) were incubated in media supplemented with radioactive glucose and insulin (1.0 mU/ml) to measure the metabolic response of the tissue to insulin. Other slices were incubated in medium supplemented with norepinephrine bitartrate (1 microgram/ml) to measure lipolytic response by glycerol release. Basal glucose utilization for oxidation, total lipid and fatty acid synthesis was higher in decap adipose tissue than intact adipose tissue. Cauterized and intact fetuses did not differ in adipose tissue glucose metabolism. Only decap adipose tissue demonstrated an insulin stimulation of glucose oxidation and lipogenesis. Norepinephrine stimulated lipolysis in both cauterized and intact adipose tissue but had no effect upon decap adipose tissue lipolysis. These results demonstrate central endocrine regulation but not central neural regulation has an important function in the development of porcine fetal adipose tissue metabolism and its responses to systemic hormones.

The function of glucocorticoids in the differentiation of porcine preadipocytes was examined. Stromal-vascular cell cultures (containing preadipocytes) derived from adipose tissue of the perirenal, ham and shoulder regions of neonatal pigs were incubated in the presence of hydrocortisone at 0 to 100 ng/ml medium. Perirenal cells did not respond to hydrocortisone with an increase in enzyme expression, nor did they demonstrate growth characteristics similar to those of cultures derived from the ham or shoulder. Cultures from the shoulder and ham regions demonstrated dose-responsive increases in enzymatic expression to hydrocortisone. Enzymatic responses by cultures derived from the ham region were lower than responses by cultures from the shoulder region as measured by changes in the activities of sn-glycerol-3-phosphate dehydrogenase and lipoprotein lipase. Addition of insulin to the medium did not produce a synergistic effect with glucocorticoid on differentiation as determined by these enzymatic parameters. However, [14C]glucose metabolism by the cells in culture was synergistically increased by insulin and glucocorticoid supplementation of the medium. The ability of hydrocortisone to induce differentiation of porcine preadipocytes in vitro suggests that the changes that occur in plasma glucocorticoid concentrations during late gestation may play an important role in the rapid development of s.c. adipose tissue in the fetal pig. Secondly, the differences in culture characteristics and hormone responses of cells derived from different locations of adipose tissue formation indicate that differences may exist in the regulation of the growth and development of preadipocytes from different anatomical locations.

Growth hormone and thyroid hormones have been implicated as important serum factors for adipocyte development in cell culture. Fetal decapitation removes these factors from serum of the growing fetal pig and results in development of fewer adipocytes than in intact fetuses. These experiments examined the effects of growth hormone or thyroxine supplementation to decapitated fetal pig sera upon pre-adipocyte proliferation and differentiation. Hormones were supplemented to concentrations present in sera from intact pig littermates (reference). Sera +/- hormones were analyzed for their effects upon pre-adipocyte proliferation as determined by [3H]-thymidine incorporation; enzyme expression as determined by sn-glycerol-3-phosphate dehydrogenase activity; and induction of complete differentiation into lipid filled adipocytes as based upon a pre-adipocyte proliferation and enzyme expression than reference sera. Growth hormone had no effect in decap sera upon these parameters. Decap sera permitted detection of 54% more lipid-accumulating, newly formed adipocytes on percol gradients than reference sera, but growth hormone reduced detection to 29% of reference sera. Thyroxine specifically stimulated pre-adipocyte proliferation more than decap sera, but not to the level of reference sera. Complete differentiation, a formation of lipid-accumulating adipocytes was promoted also by thyroxine in comparison to basal decap sera. The results of these experiments indicate thyroid hormones are an important component of fetal sera for regulation of adipocyte development, whereas growth hormone may only affect cellular metabolism and not promote pre-adipocyte growth and development.

The function of glucocorticoids in the differentiation of porcine preadipocytes was examined. Stromal-vascular cell cultures (containing preadipocytes) derived from adipose tissue of the perirenal, ham and shoulder regions of neonatal pigs were incubated in the presence of hydrocortisone at 0 to 100 ng/ml medium. Perirenal cells did not respond to hydrocortisone with an increase in enzyme expression, nor did they demonstrate growth characteristics similar to those of cultures derived from the ham or shoulder. Cultures from the shoulder and ham regions demonstrated dose-responsive increases in enzymatic expression to hydrocortisone. Enzymatic responses by cultures derived from the ham region were lower than responses by cultures from the shoulder region as measured by changes in the activities of sn-glycerol-3-phosphate dehydrogenase and lipoprotein lipase. Addition of insulin to the medium did not produce a synergistic effect with glucocorticoid on differentiation as determined by these enzymatic parameters. However, [14C]glucose metabolism by the cells in culture was synergistically increased by insulin and glucocorticoid supplementation of the medium. The ability of hydrocortisone to induce differentiation of porcine preadipocytes in vitro suggests that the changes that occur in plasma glucocorticoid concentrations during late gestation may play an important role in the rapid development of s.c. adipose tissue in the fetal pig. Secondly, the differences in culture characteristics and hormone responses of cells derived from different locations of adipose tissue formation indicate that differences may exist in the regulation of the growth and development of preadipocytes from different anatomical locations.

The role of insulin-like growth factor I (IGF-1) in the development of the porcine preadipocyte was studied. Primary cultures of stromal-vascular cells (containing preadipocytes) were derived from s.c. adipose tissue of pigs at 1 d of age by enzyme digestion and centrifugation. Cells were cultured for a total of 15 d. Cells were exposed to IGF-1 at concentrations of 0, 5, 25 or 50 ng/ml medium during one of four time periods: d 1-15, d 1-5, d 13-15, or 4 h on d 15 of culture. IGF-1 had a mitogenic effect on cells during the first three time periods as determined by coulter counting. IGF-1 induced the enzymatic differentiation of porcine preadipocytes following exposure for either the entire 15 d of culture or for only 48 h (d 13-15) after confluency had been attained (d 5). Histochemically, lipid accumulation over time paralleled changes in enzyme activity. Incubation of IGF-1 with the cell cultures during the logarithmic phase of growth (d 1-5) or for 4 h on d 15 did not affect enzyme activity. These data indicate that IGF-1 can induce the differentiation of porcine preadipocytes after the cells leave the logarithmic phase of growth through action on post-confluent events.