T.G. Ramsay

Hormonal regulation of postnatal chicken preadipocyte differentiation in vitro

This study was designed to develop a culture system from the stromal-vascular fraction of chicken adipose tissue that can be used to characterize hormones that promote preadipocyte differentiation. Abdominal adipose tissue was excised from 2 to 4-week-old male broilers (Gallus domesticus) by sterile dissection. The stromal-vascular cell fraction from the adipose tissue was isolated by collagenase digestion, filtration, and subsequent centrifugation. These preadipocytes were seeded in six well culture plates and proliferated to confluency in 10% fetal bovine serum in DMEM/F12 (50:50) medium. At confluency, experiments were initiated to determine hormonal requirements for differentiation. Insulin (100 nM) stimulated expression of citrate lyase and sn-glycerol-3-phosphate dehydrogenase relative to lactate dehydrogenase in the presence of 2.5% chicken serum (P<0.05), but not with 10% chicken serum (P>0.05). Triiodothyronine (T(3), 1 nM) and insulin-like growth factor 1 (100 ng/ml) had no effect on differentiation. Dexamethasone (Dex, 1 microM) stimulated differentiation in 2.5 or 10% chicken serum (P<0.05). Insulin, Dex and 2.5% chicken serum stimulated enzymatic differentiation to the extent of 10% chicken serum, but heparin (10 U/ml) addition, in combination with insulin and Dex was necessary to stimulate lipid filling of adipocytes.

Identification of protein carbonyls in serum of the fetal and neonatal pig

Oxidation of serum proteins leads to non-reversible carbonyl formation which alters their function and is associated with stress-related disease processes. The primary objective of this study was to quantify and identify oxidized serum proteins in fetal and newborn piglets. Protein carbonyls were converted to hydrazones with dinitrophenyl hydrazine and quantified spectrophotometrically. For identification, serum protein carbonyls were derivatized with biotin hydrazide, separated by 2D PAGE and stained with FITC-avidin. Biotin-labeled proteins were excised from gels and identified by mass spectrometry. At birth, carbonyls were determined to be approximately 600 pmole/mg serum protein. Fetuses at 50 and 100 days of gestation had similar levels of protein carbonyls as newborns. Carbonyl levels were also similar for control and runt (<1 kg at birth) piglets between 1 and 21 days of age; however, distribution of many proteins varied by age and was also influenced by birth weight. Major oxidized proteins identified in fetal (f) and newborn (n) pigs included; albumin (f, n), transferrin (f, n), fetuin-A (f, n) alpha fetoprotein (f, n), plasminogen (f, n), fetuin-B (f), alpha-1-antitrypsin (f, n) alpha-1-acid glycoprotein (f) and immunoglobulins (n). While abundance and distribution of oxidized proteins changed over time, these changes appear to primarily reflect relative amounts of those proteins in serum.

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).

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.

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.

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 (Williams 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.

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.

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.

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

This study was designed to determine whether methyl-β-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).