Domestic Animal Endocrinology 52 (2015) 51–59

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Regulation of alpha-1 acid glycoprotein synthesis by porcine hepatocytes in monolayer culture T.J. Caperna*, A.E. Shannon, M. Stoll, L.A. Blomberg, T.G. Ramsay Animal Biosciences and Biotechnology Laboratory, Beltsville Agricultural Research Center, USDA, Agricultural Research Service, Beltsville, MD 20705, USA

a r t i c l e i n f o

a b s t r a c t

Article history: Received 29 September 2014 Received in revised form 27 February 2015 Accepted 28 February 2015

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-a (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. Published by Elsevier Inc.

Keywords: Haptoglobin Tumor necrosis factor-a Oncostatin M IL-6 IL-1 IL-17a

1. Introduction Alpha-1 acid glycoprotein (AGP) is a unique hepatic acute-phase protein in the domestic pig as it is highly expressed in the preterm fetus, and after birth, it is associated with both positive and negative acute-phase responses. The concentrations of positive acute-phase

Mention of trade name, proprietary product, or vendor does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture or imply its approval to the exclusion of other products or vendors that also may be suitable. * Corresponding author. Tel.: þ1 301 504 8506; fax: þ1 301 504 8623. E-mail address: [email protected] (T.J. Caperna). 0739-7240/$ – see front matter Published by Elsevier Inc. http://dx.doi.org/10.1016/j.domaniend.2015.02.002

proteins are elevated after infection or stress, whereas negative acute-phase proteins tend to decrease after similar inflammatory challenges. Early studies suggested that AGP was elevated after bacterial infection and could be used to monitor herd health [1], whereas more recent direct evidence [2] has demonstrated a decrease in AGP expression and circulating concentration after experimental bacterial infection. In addition, AGP was not elevated in response to classic turpentine insult in pigs [3,4]. In pigs, hepatic expression of AGP has been shown to increase throughout fetal development [5] and is the major circulating serum protein at birth, and it declines dramatically thereafter [6,7]. This pattern of expression and the association of initial plasma concentration with preweaning growth rate led us

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to speculate that variability in AGP concentration within a litter may be reflective of fetal maturity as regulated within each individual placenta [8]. Many acute-phase proteins have specific functions that can attenuate or potentiate the consequences of the inflammatory response. For example, haptoglobin (HP) binds to extracellular hemoglobin, whereas the broad range protease inhibitor, alpha 2-macroglobulin, can also act as a cytokine carrier [9,10]. A clearly defined molecular function for AGP in the inflammatory process has not been ascribed [11]. In contrast, other biological functions of AGP in humans, rodents, and cattle have been recently identified; these include involvement in regulation of neutrophil degranulation, platelet aggregation, phagocytosis, cytokine induction, angiogenesis, fibroblast proliferation, and energy metabolism [12–16]. We have also recently determined that AGP inhibits the expression of lipogenic enzymes and lipogenesis in adipose tissue explants prepared from preweaned pigs (Ramsay and Caperna, article in prep). In addition, AGP is a lipocalin, and as such, is associated with the binding of many basic and hydrophobic biological and therapeutic molecules [11]. The regulation of hepatic expression of positive acutephase proteins by cytokines in human and rodent systems has been well documented and reviewed [9,10]; however, few studies have been published defining the regulation of these proteins in the pig hepatocyte [17–19]. Specifically, hepatocellular expression of AGP has not been previously reported. The primary focus of the present study was to investigate the control of AGP expression by cytokines in hepatocytes prepared from neonatal piglets and to compare its regulation with that of HP, a positive acute-phase reactant in the pig. Moreover, we also wanted to elucidate the possible roles of other metabolic hormones (insulin, glucagon, and somatotropin), as well as triiodothyronine (T3), dexamethasone (DEX), and several therapeutic reagents used to probe/modify redox and inflammatory processes in the induction and/or inhibition of AGP and HP expression.

2. Materials and methods 2.1. Hepatocyte isolation Care and treatment of all pigs in this study were approved by the Institutional Animal Care and Use Committee of the U.S. Department of Agriculture. Eight suckling crossbred female piglets from 7 different litters (5–22 d of age) were euthanized with a solution containing ketamine, telazol, xylazine, and butorphanol. Laparotomy was performed, and livers were immediately excised; the left lateral lobe was removed, the portal vein was cannulated, and hepatocytes were isolated by a 2-step collagenase digestion procedure as previously described [20] and modified [18]. Cell preparation was performed from 1 pig per week where 4 piglets were used for all RNA expression experiments and a different set of 4 piglets were used to harvest media samples for analyses of protein secretion and total cell protein. Viability of hepatocyte preparations was 80.4  0.8% by trypan blue dye exclusion (n ¼ 8).

2.2. Hepatocyte culture Hepatocytes (4.5  106) were seeded into vented T-25 flasks (Corning Inc, Corning, NY, USA) precoated with bovine skin collagen (A10644-01, Life Technologies, Grand Island, NY, USA) and were maintained in William’s E medium as previously described [18]. Except where noted, all media, cell culture, and assay reagents were purchased from Sigma (St Louis, MO, USA). Recombinant cytokines used were human oncostatin M (OSM) and porcine interleukins (ILs) (tumor necrosis factor-a [TNF], IL-6, IL-1b [IL-1]; R&D Systems, Minneapolis, MN, USA), porcine IL-17a (Kingfisher Biotech, St. Paul, MN, USA), quercetin (Calbiochem, San Diego, CA, USA), and porcine growth hormone (pGH, somatotropin), a gift from the National Pituitary Agency. Basal Williams’ E medium was prepared with 0.1 mM b-mercaptoethanol, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 10 nM Na2SeO3, 2 mM glutamine, and antibiotics (gentamicin, penicillin, streptomycin, and amphotericin B). Cells were initially maintained in basal medium containing insulin-transferrin-selenium supplement and 10% fetal bovine serum (Atlanta Biologicals, Flowery Branch, GA, USA). After a 3-h attachment period, flasks were washed with warm HEPES-buffered saline (HBS) and replaced with the same basal medium containing 5% fetal bovine serum with insulin, transferrin, and selenium. On the following day, flasks were washed twice with warm HBS, and serum-containing medium was replaced with 4.5 mL serum-free basal medium containing 1-mM carnitine, 0.01% dimethyl sulfoxide, 0.1% bovine serum albumin (BSA, A1595) 10-nM DEX, and 1 ng/mL bovine insulin (control medium). The final incubation medium for all cultures contained 0.01% BSA. Experimental conditions were established in duplicate flasks, and all experiments were performed with cells from 4 different pigs; therefore, n ¼ 4 for each treatment. The concentrations of reagents used to establish experimental conditions were as follows: all cytokines (20 ng/mL); high insulin, glucagon, and pGH (100 ng/mL); T3 (107M); high DEX (106M); hemin (50 mM); quercetin and resveratrol (100 mM); N-acetyl cysteine; and L-buthionine sulfoximine (1 mM). For protein secretion determinations, experimental conditions were established when serum-free medium was added (at 24 h), and conditioned medium was collected between 48 and 72 h. For RNA determination studies, experimental conditions were established 24 h after serum-free (control) conditions were initiated to eliminate potential serum effects. All media were changed daily and all cultures were terminated approximately 72 h after the initiation of culture, except for 4-h RNA samples which were terminated approximately 52 h after isolation of the cells. At termination, cell monolayers were washed twice with ice-cold glucose-free HBS. For the preparation of RNA, 2.0 mL TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was added to each flask on ice, and the cell material was immediately scraped and stored at 80 C. For the collection of conditioned media and cell protein, medium from each flask was aspirated and each monolayer was washed twice (as previously mentioned) and incubated with 1.4 mL mammalian protein extraction reagent (M-PER, Thermo

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Fisher Scientific, Rockford, IL, USA) with gentle rocking for 5 min at 4 C. Solubilized cell material containing cytosol and membrane proteins [18] was aspirated, and duplicate aliquots were removed for total protein analysis; the remainder was stored at 80 C. The extraction reagent contained protease and phosphatase inhibitors (P-8340, P-0044, and P-5726). Conditioned media samples were centrifuged at 3,000g, and supernatants were frozen at 80 C until analyzed. Concentration of protein in cell samples was determined by a modified Lowry method [21] after precipitation in 7% trichloroacetic acid and 0.07% Triton-X 100; BSA was used as a standard.

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Specificity of real-time PCR products was further confirmed by agarose gel electrophoresis. 2.4. Quantification of gene expression At the end of the PCR run, baseline and threshold crossing values (CT) for all analyzed genes were calculated using the Bio-Rad software, and the CT values were exported to Microsoft Excel for analysis. The expression of AGP and HP standardized against the amount of cyclophilin A messenger RNA (mRNA), was calculated using the DDCT method [23,24]. Values were calculated as the mean of duplicate determinations from duplicate tissue culture flasks derived from each of 4 individual piglets.

2.3. RNA isolation and real-time polymerase chain reaction analysis of gene expression

2.5. Analysis of AGP and HP in media

Cell samples harvested in TRIzol were thawed, and total RNA was isolated using Qiagen RNeasy spin columns (Qiagen, Valencia, CA, USA) according to the manufacturer’s protocol. Integrity of RNA was assessed via agarose gel electrophoresis, and RNA concentration was determined spectrophotometrically using ultraviolet absorbance (A) at A260 and A280 measurements. The primer sets designed to span an intron and used for real-time polymerase chain reaction (PCR) were AGP (M35990) sense, 50 -ACCCCCAGTACAATGAGTCG-30 , antisense 50 -TTAACAGCAGGTCAGCAACG -30 ; HP (NM214000) sense 50 -AGAACCCAGTGGATCAGGTG-30 , antisense 50 CCTCCTGTTTCTTTCCCACA-30 ; and cyclophilin A (AY008846) sense 50 -ATGGTAACCCCACCGTCTTC-30 , antisense 50 GTTTGCCATCCAACCACTCAG-30 . The amplicon sizes for AGP, HP, and cyclophilin A were 210, 234, and 376 bases, respectively. Thermal cycling and data acquisition were performed with a Bio-Rad iCycler IQ system (Bio-Rad Laboratories Inc, Hercules, CA, USA). Reverse transcription (RT) and real-time PCR analysis were performed in a 2-tube assay as previously described [22]. RT was performed using a Superscript First-Strand Synthesis System for RT-PCR kit (Invitrogen). Master mix was made containing random hexamers (50 ng/mL), 10-mM dNTP mix, RNase-free H2O, and RNA (1 mg/mL). The RNA mix was annealed at 65 C for 5 min. A second master mix was prepared with 10X RT buffer, 25-mM MgCl2, 0.1-mM dithiothreitol, and 1.0-mL RNaseOut. This second master mix was added to the RNA mix and incubated at 25 C for 2 min. Superscript II was then added and incubated at 25 C for 10 min, 42 C for 50 min, and 70 C for 15 min. An aliquot of RNase H (1.0 mL) was then added and incubated at 37 C for 20 min. Real-time PCR was performed using the IQ Sybr green supermix kit (Bio-Rad Laboratories Inc). A reaction mix (24 mL) was made containing 12.5-mL Sybr green supermix, 1.0mL forward primer (10 mM), 1.0-mL reverse primer (10 mM), and 9.5-mL sterile water. This reaction mix was added to each well, followed by 1.0-mL RT product. Parameters for all reactions were as follows: 1 cycle at 95 C for 15 min (PCR activation), followed by 30 cycles, 94 C for 15 s, 58 C for 30 s, 72 C for 30 s, with a final extension at 72 C for 8 min. Melting curve analysis was performed on all real-time PCR reactions to confirm specificity of the real-time PCR products. A nontemplate control was run for every assay.

A sandwich enzyme-linked immunosorbent assay (ELISA) was developed to determine the concentration of AGP in tissue culture medium obtained from porcine hepatocyte cultures. The details of this assay will be presented elsewhere. Briefly, purified porcine AGP was purchased (Life Diagnostics Inc, West Chester, PA, USA) and used to prepare an antiserum in rabbits. The whole antiserum was prepared, affinity-purified, and a portion of the affinitypurified antibody was labeled with horseradish peroxidase by Pacific Immunology, Inc (Ramona, CA, USA). Microtiter plate wells (96 well Maxisorb; Nunc, Roskilde, Denmark) were coated overnight with purified antibody in carbonate–bicarbonate buffer, pH 9.6 at 4 C, washed with 0.05% Tween-20 in phosphate-buffered saline (PBS-T, pH 7.2; Thermo Scientific, Rockford, IL, USA), blocked with 1% BSA in PBS (Thermo Scientific), and washed with PBS-T. Standards (Life Diagnostics Inc) were diluted (3.15– 200 ng/mL) in PBS-T containing 1% BSA. Media samples were diluted (1:10 or 1:20) in the same buffer, and triplicate aliquots (samples and standards) were incubated (100 mL/well) for 1 h. After washing with PBS-T, the horseradish peroxidase–labeled antibody was added to each well and incubated. Each well was washed and incubated with tetramethylbenzidine, (TMB substrate; Bethyl Laboratories, Montgomery, TX, USA), stopped with 0.18-M H2SO4, and the absorbances at 450 and 620 nm were obtained. Except where noted, all microtiter plate incubations were performed with shaking at room temperature. A standard curve was prepared (from the A450–A620 nm difference values) between 0.315 and 20 ng AGP per well, using a PL4 log-transformed sigmoidal ELISA algorithm (PRIZM 6.0; GraphPad Software Inc, La Jolla, CA, USA), and unknowns were extrapolated from the standard curve. Media from a complete set of assay conditions from an individual experiment were analyzed in triplicate on the same plate. The interassay CV of a control AGP sample (5 ng) added to each plate was 7.4%, and the CV for all triplicate media samples averaged across all plates was 4.5% (n ¼ 10 plates). The concentration of HP in media samples was obtained using a commercial porcine-specific HP ELISA kit (KA1852; Abnova, Taipei City, Taiwan). Media samples were diluted (1:20–1:500) in the provided diluent, and the assay was performed in duplicate according to the directions

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provided by the manufacturer. All media samples from an individual piglet were analyzed on a single plate, and absorbance was read at 450 nm. Standard curves and extrapolations were performed as described previously. The interassay CV was 10.9% for a control medium sample run on each of 4 plates, and the overall CV among all media sample duplicates was 6.9%. For both AGP and HP, concentrations in media were calculated as the total of each protein secreted in 24 h per mg cell protein in each flask. 2.6. Statistical analysis To evaluate the effects of treatments on the concentrations of AGP and HP, or of mRNA expression in hepatocyte cultures, the average analytical value of each condition was normalized to the average of control (basal) cultures (1-ng/ mL insulin, 10-nM DEX) in each separate experiment, where the average experimental values for each piglet were expressed as “percent of control.” Normalized data were analyzed by 1-way analysis of variance, where planned comparisons between control and experimental cultures (nonorthogonal contrasts) were evaluated (Number Cruncher Statistical Systems, Kayesville, UT, USA). Data for HP were log-transformed before performing the analysis of variance. Treatment differences were considered significant from controls at P < 0.05, and trends were considered between P < 0.05 and P < 0.07. To compare the overall correlation between mRNA expression of each protein at 24 h with the relative concentration of each secreted protein in media after the final 24-h incubation, linear regression was performed by plotting the data on a set of X (mRNA) and Y (protein in media) axes. The slope, r, and significance values of the linear relationships were determined (PRIZM 6; GraphPad Software Inc). 3. Results In this study, the influences of cytokines (TNF, OSM, IL-1, IL-6, and IL-17a), polypeptide hormones (insulin, glucagon, and pGH), a synthetic glucocorticoid (DEX), T3, and redox/ regulatory molecules (N-acetyl cysteine, quercetin, resveratrol, hemin, and L-buthionine sulfoximine) on expression of AGP mRNA (Fig. 1A, B) and HP mRNA (Fig. 2A, B) at 4-and 24-h after treatment were determined. At 4 h, quercetin and resveratrol increased AGP expression (P < 0.05). OSM (P < 0.01) and TNF (P ¼ 0.065) were associated with a decrease in expression of AGP at 24 h. High insulin (100 ng/ mL) or pGH alone did not influence the expression of AGP mRNA at 24 h; however, in combination, a specific decrease in AGP mRNA was observed (P < 0.05). In contrast, IL-1, IL17a, and resveratrol induced the expression of AGP by 24 h (P < 0.01). The abundance of HP mRNA was elevated by OSM and IL6 by 4 h (P < 0.01), and remained elevated under these conditions at 24 h (P < 0.01). In addition, HP expression was elevated by high DEX (106M) and IL-17a at 24 h, whereas TNF decreased expression of HP (P < 0.01). Redox-associated molecules had no influence on HP gene expression at 4 h or 24 h. To determine whether changes in gene expression were also associated with differences in protein synthesis and/or

secretion, ELISA analyses for porcine AGP and HP were performed on 24-h media samples collected after 48 h of treatment. For control cultures, the concentration of secreted AGP in the medium was 2.68  0.86 mg/mg cell protein per 24 h, and total extracted cell protein was 1.39  0.18 mg per flask (n ¼ 4). The secretion of AGP (Fig. 3) was inhibited by the cytokines TNF and OSM (P < 0.01), and by T3 (P < 0.05). In contrast, the secretion of AGP was increased by IL-1 and resveratrol (P < 0.01) and by IL-17a (P < 0.05). Although the basal concentration of HP (2.32  0.52 mg/mg cell protein per 24 h) was similar to that of AGP, a different pattern of HP induction/inhibition was observed (Fig. 4) when compared with AGP. OSM and IL-6 greatly (w10-fold) induced secretion of HP (P < 0.01), which was also enhanced by glucagon, 106M DEX (P < 0.01), and by T3 (P ¼ 0.051). Of all the regulatory molecules tested, only TNF was associated with a reduction in HP production (P < 0.01). 4. Discussion The goal of this investigation was to provide the first comprehensive evaluation of the regulation of AGP expression in porcine hepatocytes and to compare its expression with HP, a positive acute-phase protein. It has been recognized for quite a while that AGP is not a classic positive acute-phase protein in pigs because of no change in its circulating concentration after turpentine stress [3,4], a treatment associated with induction of several positive acute-phase proteins, including HP. In addition, the concentrations of AGP were not correlated with other acutephase proteins in pigs during early growth [7], or in a sampling of normal older pigs [25]. Recent evidence has further suggested that AGP is indeed a negative acutephase protein [2], where circulating concentrations and hepatic gene expression were acutely depressed after specific infection with Actinobacillus and Staphylococcus bacteria. In this study, short-term (4 h) induction of AGP expression was observed with quercetin and resveratrol, but mRNA abundance returned to control values by 24 h in the presence of quercetin, with no observed effect on long-term AGP protein synthesis. The relevance of short-term upregulation of AGP mRNA, with no corresponding influence on protein synthesis, remains to be determined. OSM essentially shuts down AGP transcription by 24 h, and this reduction was highly correlated with a decline in AGP protein secretion. In addition, TNF reduced mRNA abundance and secretion of AGP. By 24 h, IL-17a, IL-1, and resveratrol treatments induced AGP mRNA abundance and protein secretion. Resveratrol is an anti-inflammatory polyphenol with potential estrogen receptor activity [26], and is associated with short- [27] and long-term effects on hepatic lipid metabolism and gene expression [28]. The potential anti-inflammatory effects of resveratrol are known to be due in part to inactivation of nuclear factor-kB [29], a key inflammatory pathway regulator. Because TNF activates nuclear factor-kB in piglet hepatocytes [18] and inhibited AGP expression here, it would be attractive to postulate that resveratrol might be acting by inhibiting nuclear factor-kB in piglet hepatocytes. However, other transcription factors must also be involved in AGP

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Fig. 1. Relative messenger RNA abundance of alpha-1 acid glycoprotein (AGP) in primary cultures of porcine hepatocytes at 4 h (A) and 24 h (B) after the establishment of experimental conditions. Cultures were incubated in serum-free basal (control) medium containing 1-ng/mL insulin (Ins) and 108-M dexamethasone (DEX) for 24 h before adding experimental treatments. Data are expressed as the percent of each condition relative to expression in controls (n ¼ 4). Experimental conditions were as follows: human oncostatin M (OSM) and porcine interleukins (ILs) (tumor necrosis factor [TNF], IL-6, IL-1, and IL-17a at 20 ng/ mL), porcine growth hormone (pGH, 100 ng/mL), Ins (100 ng/mL), glucagon (100 ng/mL), triiodothyronine (T3, 107M), DEX (106M), hemin (50 mM), quercetin and resveratrol (100 mM), N-acetyl cysteine (NAC, 1 mM), and L-buthionine sulfoximine (BSO, 1 mM). Data were analyzed by 1-way analysis of variance. *P < 0.05, **P < 0.01, and “a” for (P ¼ 0.065) compared with controls.

regulation as OSM did not activate nuclear factor-kB in piglet hepatocytes [18], but markedly downregulated AGP expression and secretion. In addition, quercetin is a polyphenol antioxidant which also has been shown to block nuclear factor-kB activation in rat hepatocytes [30], and in the present study, had no long-term influence on AGP mRNA abundance or protein secretion. We previously reported [18] that TNF alone did not influence HP synthesis/secretion in porcine hepatocytes in 24 h, whereas TNF did inhibit HP secretion in the presence of IL-6. It was also reported that TNF had no effect on

secretion of HP in porcine hepatocytes [17]; however, in that study, all cells were incubated in 10–6-M DEX which we now show here is a concentration of DEX which induces HP expression and synthesis. It would appear that TNF does not down-regulate HP expression at 24 or 48 h in the presence of high DEX [17], but does so relative to basal conditions (Fig. 4). In the present study, HP mRNA abundance was not decreased at 4 h by TNF but was lower by 24 h after TNF addition, which led to the observed decrease of HP in media on the following day, after a 48-h induction period. Our data clearly show for the first time that HP

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Fig. 2. Relative messenger RNA abundance of haptoglobin (HP) in primary cultures of porcine hepatocytes at 4 h (A) and 24 h (B) after the establishment of experimental conditions. All experimental conditions were as described for Figure 1. *P < 0.05, **P < 0.01 compared with controls. BSO, L-buthionine sulfoximine; DEX, dexamethasone; IL, interleukin; Ins, insulin; NAC, N-acetyl cysteine; OSM, oncostatin M; pGH, porcine growth hormone; TNF, tumor necrosis factor.

expression and protein secretion are downregulated by TNF in porcine hepatocytes. Growth hormone was shown to highly suppress AGP expression in the rat liver [31], and we hypothesized that because AGP was elevated in piglet runts [8] that pGH may also be associated with AGP regulation in porcine hepatocytes. However, in the present study, pGH alone did not appear to regulate AGP mRNA abundance or protein secretion, but pGH in the presence of high insulin inhibited AGP mRNA expression without altering AGP protein synthesis/secretion. Although we have previously demonstrated that porcine hepatocytes cultured under similar conditions do indeed have active GH receptors [32,33], these data suggest that at least in the pig hepatocyte, GH

does not appear to be a major regulator of AGP or HP synthesis. In the present study, in addition to resveratrol, IL-17a and IL-1 were shown for the first time to be positive regulators of AGP expression, and IL-17a also increased HP mRNA expression, but with no apparent increase in HP protein secretion. The available data on the regulation of IL17a production and its function in domestic animals is sparse. IL-17a has been shown to be elevated in gastrointestinal inflammation and other inflammatory diseases in pigs [34]. Although in mouse hepatocytes, IL-17a induced many genes including nuclear factor-kB [35], the potential influences of IL-17a on porcine hepatic gene expression were unknown until the present study.

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Fig. 3. Secretion of alpha-1 acid glycoprotein (AGP) into media from primary cultures of porcine hepatocytes in 24 h. Hepatocyte monolayers were maintained in the presence of control medium or in conditions as described in Figure 1 for 48 h, and media samples were collected during the last 24 h. At termination, media samples were collected and cell material was harvested. Cells were isolated from 4 piglets (n ¼ 4). *P < 0.05, **P < 0.01 compared with control. BSO, L-buthionine sulfoximine; DEX, dexamethasone; IL, interleukin; Ins, insulin; NAC, N-acetyl cysteine; OSM, oncostatin M; pGH, porcine growth hormone; TNF, tumor necrosis factor.

To further evaluate the correlation between overall mRNA abundance and protein secretion, data for media concentrations of both AGP and HP were plotted against

the relative mRNA values (Fig. 5). The linear correlation for both proteins was highly significant (P < 0.001), indicating that overall, the secretion of each protein is reflective of the

Fig. 4. Secretion of haptoglobin (HP) into media from primary cultures of porcine hepatocytes in 24 h. Hepatocyte monolayers were maintained in the presence of control medium or in conditions as described in Figure 1 for 48 h, and media samples were collected during the last 24 h. Media and sample preparation were as described previously (Fig. 3). *P < 0.05, **P < 0.01, and “a” (P ¼ 0.051) compared with control. BSO, L-buthionine sulfoximine; DEX, dexamethasone; IL, interleukin; Ins, insulin; NAC, N-acetyl cysteine; OSM, oncostatin M; pGH, porcine growth hormone; TNF, tumor necrosis factor.

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In summary, this is the first study to examine the regulation of AGP in porcine hepatocytes where IL-1, IL-17a, and resveratrol were shown to be positive regulators of AGP expression. We have shown here that cytokines and inflammatory regulators also have negative (TNF, OSM, and T3) effects on its expression and secretion, and that the regulation of expression of HP and AGP are indeed distinct for these 2 members of the acute-phase protein family. The role of AGP in the inflammatory response and its effects on metabolism and gene expression in other cells, including porcine adipose and intestinal cells, is currently under investigation in our laboratory. Acknowledgments Jenile Tapscott, Russ Lange, and James Woods are thanked for assistance in maintaining and handling experimental animals. Neil Talbot and Ted Elsasser are thanked for helpful discussions on cell culture and cytokine-directed metabolism. References

Fig. 5. Comparison of messenger RNA (mRNA) expression and protein secretion into media from porcine hepatocyte monolayers. Data (24 h mRNA vs secreted proteins, as percent of control) for all treatments were plotted for AGP (A) and HP (B) and each relationship was subjected to linear regression analysis as described in materials and methods.

changes (both positive and negative) elicited by the regulatory molecules at the mRNA level. Interestingly, the relationship between protein and mRNA differed between the 2 genes investigated, such that the slope for HP was 1.12  0.13, indicating that the relative changes in secreted protein were roughly equivalent to the observed change in mRNA abundance. The slope of the relationship between protein secretion and mRNA abundance for AGP was 0.42  0.06, indicating that the perturbation in transcriptional activity was approximately twice that observed for protein secretion for the various treatments. Whether this indicates possible differences in rates of protein turnover, translation, post-translational modification, or secretion for these 2 proteins, remains to be determined. Nevertheless, the data indicate strong transcriptional control with correlated expression of mRNA and protein secretion in these 2 highly, yet differentially, regulated proteins.

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