Cloning of porcine proglucagon and effect of commensal bacteria on relative gene expression in the intestine of gnotobiotic pigs

Cloning of porcine proglucagon and effect of commensal bacteria on relative gene expression in the intestine of gnotobiotic pigs Can. J. Anim. Sci. D...
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Cloning of porcine proglucagon and effect of commensal bacteria on relative gene expression in the intestine of gnotobiotic pigs

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R. H. Siggers, T. W. Shirkey, M. D. Drew, B. Laarveld, and A. G. Van Kessel1 Department of Animal and Poultry Science, 51 Campus Drive, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 5A8. Received 21 September 2007, accepted 31 March 2008. Siggers, R. H., Shirkey, T. W., Drew, M. D., Laarveld, B. and Van Kessel, A. G. 2008. Cloning of porcine proglucagon and effect of commensal bacteria on relative gene expression in the intestine of gnotobiotic pigs. Can. J. Anim. Sci. 88: 429438. Porcine proglucagon mRNA sequence was determined by designing primers based on homologous regions in bovine and human genes. The porcine sequence shared 90% nucleotide identity with human proglucagon; however, predicted Ser159Arg and Leu160Lys substitutions were observed. This newly identified sequence suggests that the intestinal trophic peptide, glucagon-like peptide 2 (GLP-2), is actually 35 amino acids in the pig rather than 33 amino acids, as previously reported. To determine the effect of different bacteria on intestinal proglucagon gene expression, 16 piglets were reared in gnotobiotic isolators maintained germ-free (GF), monoassociated with Lactobacillus fermentum (LF) or Escherichia coli (EC), or conventionalized (CV) in two separate experiments for 13 d. Cultured cecal contents confirmed microbial status with the exception of GF pigs in exp. 2, which were contaminated with Staphylococcus epidermidis (SE). Mean fold differences in ileal proglucagon expression (CV set to 1) were 1.0ab, 1.6a, 0.7b, 1.0ab for GF, LF, EC and CV in exp. 1 and 4.0a, 2.0b, 0.6c, 1.0c for SE, LF, EC and CV groups in exp. 2, respectively. This study, for the first time, accurately describes the full nucleotide sequence for porcine proglucagon, and shows that proglucagon expression is differentially regulated by bacteria colonizing intestine of neonatal piglets. Key words: Proglucagon, gnotobiotic, pig, gene expression, intestine Siggers, R. H., Shirkey, T. W., Drew, M. D., Laarveld, B. et Van Kessel, A. G. 2008. Clonage du proglucagon du porc et incidence des bacte´ries commensales sur l’expression relative des ge`nes dans l’intestin des porcs gnotobiotes. Can. J. Anim. Sci. 88: 429438. Les auteurs ont e´tabli la se´quence de l’ARNm porcin qui code le proglucagon en pre´parant des amorces d’apre`s les re´gions homologues des ge`nes bovins et humains. La se´quence porcine a 90 % de nucle´otides identiques a` ceux du proglucagon humain. Cependant, les auteurs ont note´ la substitution des nucle´otides Ser159Arg et Leu160Lys. La nouvelle se´quence laisse croire que le peptide trophique de l’intestin, le peptide 2 analogue au glucagon (GLP-2), se compose de 35 acides amine´s chez le porc au lieu de 33, comme on l’avait d’abord signale´. Pour ve´rifier l’incidence de diverses bacte´ries sur l’expression du ge`ne codant le proglucagon dans l’intestin, les auteurs ont proce´de´ a` deux expe´riences distinctes durant lesquelles ils ont e´leve´ 16 porcelets pendant 13 jours en isolement, dans des conditions abiotiques (GF) ou en pre´sence de Lactobacillus fermentum (LF) ou d’Escherichia coli (EC), ou dans des enclos classiques (CV). La culture des e´chantillons fe´caux confirme l’e´tat microbiologique sauf pour les porcelets GF de la deuxie`me expe´rience, qui e´taient contamine´s par Staphylococcus epidermidis (SE). Les diffe´rences de grandeur moyennes dans l’expression du proglucagon ile´al (CV e´gale 1) e´taient de 1,0ab, 1,6a, 0,7b et 1,0ab pour les porcelets GF, LF, EC et CV dans la premie`re expe´rience et de 1 et 4,0a, 2,0b, 0,6c et 1,0c pour les groupes SE, LF, EC et CV dans la seconde, respectivement. Cette e´tude caracte´rise pour la premie`re fois avec pre´cision la se´quence comple`te de nucle´otides du proglucagon porcin et re´ve`le que l’expression du ge`ne qui le code est re´gule´e diffe´remment par les bacte´ries qui colonisent l’intestin des porcelets nouveau-ne´s. Mots cle´s: Proglucagon, gnotobiote, porc, expression des ge`nes, intestin

In mammals, proglucagon is a preprohormone transcribed, translated, and differentially cleaved in three primary tissues including pancreatic islet cells, enteroendocrine L cells located in the distal ileum and colon, and the brain (Burrin et al. 2003). In the pancreas, the primary proglucagon-derived peptide is glucagon, a well-described regulator of glucose metabolism (Lovshin and Drucker 2000). In the gastrointestinal tract and

brain, the primary products derived from proglucagon posttranslational processing are glucagon-like peptide 1 (GLP-1) and glucagon-like peptide 2 (GLP-2) (Drucker 1999; Burrin et al. 2003). GLP-1 has multiple regulatory

Abbreviations: CT, threshold cycle; CV, conventionalized; EC, Escherichia coli; GF, germ-free; LF, Lactobacillus fermentum; GLP-1, glucagon-like peptide 1; GLP-2, glucagon-like peptide 2; PVDF, polyvinylidene diflouride; SE, Staphylococcus epidermidis; SI, small intestine

1

To whom correspondence should be addressed (e-mail: [email protected]). 429

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430 CANADIAN JOURNAL OF ANIMAL SCIENCE

properties involving appetite depression, gastric emptying, stimulation of glucose-dependant insulin secretion, and inhibition of glucagon secretion (Keiffer and Habener 1999, Lovshin and Drucker 2000). GLP-2 has been identified as an important regulator of intestinal growth, epithelial replacement rate, apoptosis, and nutrient absorption (Brubaker et al. 1997; Drucker 1999; Burrin et al. 2003). Previous studies using the neonatal gnotobiotic pig model developed in this laboratory have demonstrated that differences in microbial colonization can differentially affect intestinal morphology, protein expression, and inflammatory cytokine and chemokine gene expression (Alison and Sarraf 1994; Danielsen et al. 2007; Meurens et al. 2007). One of the most striking differences shown in the previous studies was the ability of different bacteria to significantly influence intestinal morphology. Differential regulation of proteins involved in enterocyte proliferation and migration lead to marked differences in villus height and crypt depth as well as indicators of enterocyte turnover (Chowdhury et al. 2007; Danielsen et al. 2007; Willing and Van Kessel 2007). These responses are consistent with the physiological effects of the proglucagon derived peptide, GLP-2. The pig is a model commonly used to investigate neonatal intestinal development including the bioactivity of proglucagon-derived peptides. Thus, it is surprising that the nucleotide sequence of porcine proglucagon has not previously been elucidated. As a result, the first objective of the current study was to sequence the porcine proglucagon gene. Second, we hypothesized that bacteria may differentially regulate proglucagon expression contributing to observed differences in intestinal morphology and function. Thus, using the same gnotobiotic pigs as described in a companion study (Shirkey et al. 2006), we compared the effect of different bacteria on proglucagon gene expression in the pig intestine. Identifying early regulators of intestinal growth and function in the neonatal pig may lead to strategies aimed at improving expression of these factors, leading to improved performance and health during the neonatal period. We therefore set out to investigate whether proglucagon participates in the early bacterial-induced effects on intestinal growth. MATERIALS AND METHODS Cloning of Proglucagon Alignment of bovine (accession K00107) and human (accession V01515), proglucagon nucleotide sequences (GenBank BLAST† , National Center for Biotechnology Information, Bethesda, MD) identified homologous regions just 5? and 3? to the protein encoding region of preproglucagon. Forward (5?/GGTGTCAGAAGGCAGCAAAA/3?) and reverse (5?/CATGGCTGGCA GGTGATATT/3?), PCR primers (InvitrogenTM Life Technologies, Burlington, ON) were designed on these

regions and used to amplify cDNA prepared from an adult pig pancreas and whole distal small intestine (SI) collected from three 13-d-old pigs. The 50 mL PCR reaction mixture (InvitrogenTM Life Technologies) consisted of 10 PCR Buffer (20 mM Tris-HCl pH 8.4, 50 mM KCl), 1.5 mM MgCl2,, 0.2 mM dNTP mix, 400 nM of each primer, 5 U Taq DNA polymerase, 1 mL cDNA, and ddH2O. The reaction conditions for amplification were: pre-dwell for 4 min at 958C, 40 cycles of denaturing for 1 min at 958C and annealing for 30 s at 57.18C, and a final extension for 1 min at 728C. The PCR products were separated on agarose gel, gene cleaned (Geneclean II, Bio 101, Carlsbad, CA), and cloned (TOPO TA Cloning† Kit, Invitrogen† , CA) prior to sequencing (Plant Biotechnology Institute, Saskatoon, SK). Nucleotide sequences were compared using SequencerTM (Version 4.05, Gene Codes Corporation, Ann Arbor, MI) and submitted to GenBank (accession AY242124). Derivation and Rearing of Gnotobiotic Pigs Gnotobiotic isolators were prepared and piglets were reared as previously described in a companion study (Shirkey et al. 2006). Briefly, 16 piglets (800 g BW, Large White White Duroc) were aseptically obtained from two sows via Caesarean section (114 d gestation), passed through a betadine dip tank and revived in a sterile HEPA-filtered transfer unit. Piglets were transported in the transfer unit to the gnotobiotic facility and aseptically allocated into four gnotobiotic isolators balanced for litter of origin and sex. Piglets were bottle-fed sterile-filtered porcine serum (Gibco, Burlington, ON) mixed with Similac† (Abbott Laboratories, Abbott Park, IL) in a 1:1 (vol/vol) ratio ad libitum immediately following revival until 24 h of age. Thereafter, piglets were adjusted to trough-feeding and fed ad libitum a mixture of 2:1 Similac† :water (vol/vol) three times daily. The last feeding occurred at 2400 on day 12. Isolator temperature was 348C at day 0 and decreased to 318C by day 13. All procedures involving animals were in accordance with guidelines defined by the Canadian Council on Animal Care. Treatment Assignment and Experimental Design Two experiments of identical design and employing 16 piglets assigned to four isolator units were conducted. In each experiment, at 24 h and 30 h post-partum, piglets in three isolators were orally inoculated with an overnight culture of porcine cecal isolates of either non-pathogenic Gram-negative Escherichia coli (EC, 109 cfu mL 1), Gram-positive Lactobacillus fermentum (LF, 108 cfu mL 1), or with fresh adult porcine feces plus EC and LF (CV). Fresh fecal material was obtained from sows in a research swine herd (Prairie Swine Centre Inc., Saskatoon, SK) and made into a slurry containing 50% feces and 50% sterile phosphate-buffered saline. Two milliliters of LF and EC overnight cultures were added to 2 mL of fecal slurry (TV 6 mL) for the CV

SIGGERS ET AL. * PORCINE PROGLUCAGON AND MICROBIAL EFFECTS ON EXPRESSION

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inoculant. LF and EC pigs were orally administered 2 mL of inoculant, and CV pigs were orally administered 6 mL of inoculant. One isolator was maintained germfree (GF). In exp. 2, cecal cultures confirmed that the GF isolator was contaminated. Clinical typing at Prairie Diagnostic Services (Saskatoon, SK) identified the contaminant as a single strain of Staphylococcus epidermidis (SE). As a result this group was designated SE in subsequent analysis and discussion. Tissue Collection On day 13, piglets were removed from the isolators and BW recorded. After immersion in CO2 and exsanguination, cecal contents were aseptically collected to confirm microbial status. The SI was removed and length was recorded. One 30-cm tissue segment was taken from each location corresponding to 5, 25, 50, 75, and 95% of SI length, beginning at the pyloric sphincter. Contents were rinsed from intestinal segments using cold sterile phosphate-buffered saline. Two 10-cm segments were excised from each segment, snap frozen in liquid N2 and stored at 808C. Microbial Identification and Enumeration Brain heart infusion broth with 0.5% cysteine hydrochloride was sealed and sterilized in glass tubes containing sterile swabs, and placed in the GF isolator prior to peracetic acid sterilization. Swabs were wiped perianally and placed back into the culture broth daily from days 0 to 4 and every second day thereafter. The absence of visible bacterial growth confirmed bacteriafree status. On day 13, cecal contents (12 g) from each piglet were diluted to 10 2 and 104 in peptone water, and plated in duplicate using the Autoplate† 4000 (Spiral Biotech Inc., Bethesda, MD). Diluted contents from all pigs were plated on blood agar base (Sparks, MD) with 5% defibrinated sheep blood and cultured under aerobic and anaerobic (GasPak† anaerobic system, Becton Dickinson microbiology system, Franklin Lakes, NJ) conditions, for enumeration of total aerobes and anaerobes, respectively. Contents from LF and CV pigs were plated on MRS agar (blood agar base) and incubated under anaerobic conditions. Contents from EC and CV were plated on MacConkey agar (Difco Laboratories) and incubated under aerobic conditions. All plates were incubated at 378C for 24 48 h prior to colony enumeration. Colony isolates that differed morphologically from the inoculant strains were isolated and submitted to Prairie Diagnostic Services for clinical typing. In exp. 2 after confirmation of a Staphylococcus contamination of the GF treatment group, frozen digesta (808C) from LF and EC treatment groups was cultured on Mannitol salt agar (Difco Laboratories) to evaluate potential cross contamination of Staphylococcus spp. to other treatment groups.

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RNA Isolation and cDNA Synthesis A 10-cm segment of frozen intestinal sample at each location was pulverized with a mortar and pestle under liquid N2. Total RNA was extracted from approximately 200 mg of pulverized tissue using a commercial kit (RNeasy Mini Kit; Qiagen, Cat. No. 74104). Total RNA was quantified by optical density at 260 nm and first strand cDNA was generated (Invitrogen SuperscriptTM First-Strand Synthesis System for RT-PCR, Cat. No. 11904018) from 1 mg total RNA using Oligo dT primers. Relative Quantification of Proglucagon Transcripts Based on the porcine proglucagon nucleotide sequence reported here and intron/exon boundaries deduced by comparison with the human proglucagon gene, intron spanning PCR primers (forward, 5?/ATGACTGAAGACAAGCGCCACT/3?; reverse, 5?/TCATCG TGACGTTTGGCAATGT/3?; InvitrogenTM Life Technologies) and a Taqman† probe (5?6-FAMTM/TCTTGTTCCTCTTGGTGTTCATCAGCCACT/3? BHQTM, Integrated Technologies, Inc., Coralville, IA) were designed for quantification of proglucagon transcripts relative to porcine glyceraldehyde phosphate dehydrogenase (pGAPDH) using quantitative real-time PCR (qPCR). pGAPDH was quantified using intron-spanning forward (5?/GTTTGTGATGGGCGTGAAC/3?) and reverse (5?/ATGGACCTGGGTCATGA GT/3?) primers and a Taqman† probe (5?6-FAM TM/CTCCACGATGCCGAAGTGGT/3? BHQTM) reported previously by Shirkey et al.(2006). qPCR primers amplified products of the expected size when using cDNA prepared from pig pancreas as template. No PCR product was observed when cDNA was prepared without reverse transcriptase (No-RT control). In five separate experiments, duplicate qPCR amplifications of proglucagon and pGAPDH were conducted using a twofold dilution series of cDNA prepared from porcine pancreas as template in a 25-mL reaction mix [0.75 U Platinum† Taq DNA polymerase, 10 mM TrisHCl (ph 8.4), 25 mM KCl, 1.5 mM MgCl2, 100 mM dGTP, 100 mM dATP, 100 mM dCTP, 200 mM dUTP, 0.5 U UDG, and stabilizers (Platinum† Quantitative PCR SuperMix-UDG, Invitrogen, Carlsbad, CA), 400 nM forward primer, 400 nM reverse primer, 200 nM Taqman† probe]. PCR amplification was performed using the iCyler iQ Real-Time PCR detection system (Bio-Rad, Hercules, CA). Amplification conditions for pGAPDH and proglucagon included a pre-dwell for 4 min at 958C and 40 cycles of denaturing for 1 min at 958C and annealing for 30 s at 60.08C. The threshold cycle (CT) for proglucagon and pGAPDH was determine for each cDNA dilution using iCyclerTM iQ Optical System Software (Version 3.0a, Bio-Rad, Hercules, CA) and the DCT (CT(GAPDH)  CT(proglucagon)) calculated. The DCT for each dilution was plotted against the log of total cDNA volume to confirm equivalent PCR

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432 CANADIAN JOURNAL OF ANIMAL SCIENCE

efficiency (absolute value of slope B0.1) for pGAPDH and proglucagon (data not shown) allowing application of the 2DDCT equation to calculate mean fold change in expression (Livak and Schmittgen 2001). For quantification of proglucagon expression in intestine of gnotobiotic pigs the mean CT value for pGAPDH and proglucagon was determined from triplicate qPCR assays using cDNA prepared from SI tissue collected at 50, 75 and 95% of SI length. The DCT values were calculated and 2DDCT methodology was used to determine mean fold-change in proglucagon expression relative to CV pigs across treatments and relative to the 50% intestinal location within treatment. Caspase-3 Protein Western Blot Assay Small intestinal frozen tissue collected at 75% of intestinal length were homogenized and pooled according to location within treatment. Pooled tissue homogenates (15 mg of protein) were analyzed for protein concentration using the Bio-Rad protein microassay procedure (Bio-Rad Laboratories), and were separated by molecular weight using SDS-PAGE (Assorted Ready Gel† Precast Gels, 18% acrylamide, Bio-Rad). Human caspase-3 (active subunit 17 kDa) recombinant protein (Catalog number: CC119, Chemicon International, Temecula, CA) was included in a separate lane as a positive control. Separated protein was transferred to polyvinylidene diflouride (PVDF) membranes (Immuno-BlotTM PVDF Membrane-0.2 mm, Bio-Rad) using the Mini Trans-Blot† Electrophoretic Transfer Cell and the recommended protocol (Bio-Rad). PVDF membrane blocking and immunoblotting was performed following Opti-4CNTM Substrate Kit protocol (Goatanti-Rabbit, Bio-Rad). Specific reagents used included: 5% non-fat dry milk (Blotting Grade Non-Fat Dry Milk, Bio-Rad) for blocking; rabbit-anti-active caspase3 polyclonal antibody (Catalog # AB3623, Chemicon International, Temecula, CA) diluted 1:2000 goat-antirabbit secondary antibody conjugated with horseradish peroxidase (Opti-4CNTM Substrate Kit) diluted 1:8000. Statistical Analysis Each experiment was analyzed as a two-way ANOVA using the general linear model procedure (SPSS program, Chicago, IL). All isolators were maintained identically and housed in the same room in each experiment such that piglet was considered the experimental unit. Sources of variation included microbial treatment and intestinal location. For qPCR data, statistical differences are based on statistical analysis of DCT values. Means were separated by Ryan-EinotGabriel-Welsch multiple F test. The level of significance used was P B0.05.

RESULTS Cloning of Proglucagon Amplification of pig pancreas cDNA using proglucagon primers designed for homologous regions in human and bovine proglucagon resulted in a PCR product of approximately 630 bp, as expected. Sequencing of three different plasmid clones containing the PCR product yielded identical sequence over the entire putative protein coding sequence. Sequencing of PCR product amplified from three different samples of porcine ileal cDNA prepared from three 13 d-old pigs matched identically with the pancreas derived nucleotide sequence. High nucleotide homology sequence homology was observed among the porcine proglucagon coding region (accession AY242124) and human (accession X03991), bovine (accession K00107), and mouse (accession Z46845) proglucagon coding sequences (Fig. 1). The human nucleotide and predicted amino acid sequence shared 90 and 91% sequence identity with porcine proglucagon, respectively. A single localized region of nucleotide sequence variation was observed at the carboxy-terminus of proglucagon predicting amino acids serine and leucine in the pig sequence versus arginine and lysine in human, bovine and mouse sequences at position 159 and 160, respectively. The predicted porcine amino acid sequence contrasts previous work using peptide purification (Buhl et al. 1988) and epitope mapping ( Ørskov et al. 1989) suggesting that arginine and lysine are located at the carboxyterminal positions for porcine proglucagon, as observed in humans (Fig. 2). Microbial Status of Gnotobiotic Pigs As reported by Shirkey et al. (2006) in exp. 1, culture of cecal contents from GF pigs resulted in no bacterial growth. In exp. 2, 3.5 108 cfu g1 S. epidermidis was detected in cecal contents of GF pigs. In both experiments, culture of cecal contents from LF and EC pigs indicated monoassociation with L. fermentum (exp. 1, 6.1 107 cfu g1; exp. 2, 4.9 107 cfu g1) and E. coli (exp. 1, 1.7 109 cfu g 1; exp. 2 3.5 109 cfu g1), respectively. For exp. 2, cross contamination of other treatments with SE was excluded by the lack of colony growth on mannitol salt agar. Culture of cecal contents from CV pigs on blood agar resulted in numerous morphologically distinct colonies under aerobic (exp. 1, 1.4 109 cfu g1; exp. 2, 2.2 109 cfu g1) and anaerobic (exp. 1, 1.9 109 cfu g1; exp. 2 3.4 109 cfu g1) conditions. Health Status As reported by Shirkey et al. (2006), in exp. 1, all 16 pigs were included in experimental analysis. In exp. 2, one pig from the CV group was emaciated by the end of the experiment and was excluded from experimental analysis. All other pigs in exps. 1 and 2 appeared healthy, as

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SIGGERS ET AL. * PORCINE PROGLUCAGON AND MICROBIAL EFFECTS ON EXPRESSION Porcine Human Bovine Mouse

: : : :

ATGAAAACCATTTACTTTGTGGCTGGATTGTTTGTAATGCTGGTACAAGGCAGCTGG .......G.....................A........................... .......G.C............................................... .....G........................C..A..........G............

: : : :

57 57 57 57

Porcine Human Bovine Mouse

: : : :

CAACGTTCCCTTCAGAATACAGAGGAGAAATCCAGATCATTCCCAGCTCCCCAGACG ..............AG.C........................T.....T.....G.A .................C.................T....................C ..G.ACG.......AG.C...........CC.................T.......A

: : : :

114 114 114 114

Porcine Human Bovine Mouse

: : : :

GACCCTCTCGATGATCCAGATCAGATGACTGAAGACAAGCGCCACTCACAGGGCACG .....A...AG......T..........AC..G...........T...........A .....G....GC..............C.A......T...........G........A ..AG.G.AT..G..C..T...G......A.........A.................A

: : : :

171 171 171 171

Porcine Human Bovine Mouse

: : : :

TTTACCAGTGACTACAGCAAGTATCTGGACTCCAGGCGTGCCCAGGATTTTGTGCAG ..C.........................................A............ ..C....................C.......................C..C...... ..C.....C...........A..C..C......C.C........A............

: : : :

228 228 228 228

Porcine Human Bovine Mouse

: : : :

TGGCTGATGAACACCAAGAGGAACAAGAATAACATTGCCAAACGTCACGATGAATTT ...T.......T.............G............................... ...T.......T........A..........................T......... ...T....................CG...C.................T.........

: : : :

285 285 285 285

Porcine Human Bovine Mouse

: : : :

GAGAGACATGCTGAAGGGACCTTTACCAGTGATGTAAGTTCTTATTTGGAAGGCCAA ......................................................... ......................................................... ...................................G........C.....G.....G

: : : :

342 342 342 342

Porcine Human Bovine Mouse

: : : :

GCTGCCAAGGAATTCATTGCTTGGCTGGTGAAAGGCCGAGGAAGGCGAGATTTCCCA ......................................................... ......................................................... ..A..A..............G.............................C......

: : : :

399 399 399 399

Porcine Human Bovine Mouse

: : : :

GAGGAAGTTACCATTGTCGAAGAACTCCGCCGCAGACACGCTGATGGCTCCTTCTCA ..A..G..CG.......T........TG..........T........T..T.....T ..A.....C.A...C..T.......................C........T.....T ..A.....CG......CT..G......G.......G....................T

: : : :

456 456 456 456

Porcine Human Bovine Mouse

: : : :

GATGAAATGAACACTGTTCTCGATAATCTTGCCACCCGAGACTTTATAAATTGGCTC .....G........CA....T............G..A.G...........C...T.G .....G...................G........................C...T.G .....G....G...CA....G..C............A.G.....C..C..C.....G

: : : :

513 513 513 513

Porcine Human Bovine Mouse

: : : :

CTTCACACCAAAATTACTGACAGTCTCTAGGAAGTATGTCACTAATCAAGATCGTGT A....G........C........GTGA-----------------------------.....G..G..............GAAG..A--------------------------A....A.....G..C.......AGAAA...---------------------------

: : : :

570 540 543 543

433

Fig. 1. Nucleotide alignment of the protein coding region for porcine (accession AY242124), human (accession X03991), bovine (accession K00107), and mouse (accession Z46845) proglucagon. 5?ATG (start codon) and 3?TAG (stop codon) for porcine proglucagon are shaded. Dots indicate nucleotide identity with porcine sequence and dashes indicate the corresponding nucleotide was not used in the alignment.

indicated by mean treatment body weights. Mean body weights (9SE) on day 13 for GF, LF, EC and CV pigs (exp. 1) were 2.82 kg90.1, 3.24 kg90.31, 2.62 kg90.02,

and 2.89 kg90.21, respectively. Mean body weights (9SE) on day 13 for SE, LF, EC and CV pigs (exp. 2) were 3.15 kg90.48, 3.18 kg90.38, 2.89 kg90.13, and

434 CANADIAN JOURNAL OF ANIMAL SCIENCE GLP-2

VAL

ARG

SER LEU

PREDICTED PORCINE

LEU HIS

THR

3

LEU HIS

PORCINE

THR

ALA ILE VAL GLU GLU LEU GLY ARG ARG HIS ALA ASP GLY SER PHE SER ASP GLU MET ASN THR ILE LEU ASP ASN LEU ALA ALA ARG ASP PHE ILE ASN TRP LEU ILE GLN THR LYS ILE THR ASP ARG LYS

2

THR

HUMAN

VAL

1

THR

160

Fig. 2. Alignment of the predicted amino acid sequence of the carboxy-terminus of human proglucagon (row one) (GenBank accession X03991) and the deduced sequence of carboxy-terminal porcine proglucagon (row two) determined by peptide purification and epitope mapping (Buhl et al. 1988; Ørskov et al. 1989). Row three shows the predicted porcine amino acid sequence based on the nucleotide sequence reported here. The proglucagon region corresponding to GLP-2 is indicated above the alignment. Regions homologous with the human sequence are indicated by a dashed line.

2.61 kg90.36, respectively. Isolator reared pigs had body weights slightly lower than typically seen for sow reared pigs (Jones et al. 2002). Validation of Proglucagon qPCR Primers and Probe An expected 145 bp PCR product was amplified using real-time PCR primers to amplify cDNA prepared from ileum of two 13-d-old pigs. The absence of PCR product for the No-RT control indicated that the primers did not amplify genomic cDNA under the conditions employed (data not shown). A plot of the log of total cDNA volume used in qPCR versus DCT, calculated by subtracting the CT for proglucagon from the CT for pGAPDH was generated. The absolute slope of the regression line was 0.0469 indicating similar PCR efficiency for pGAPDH and proglucagon and validating use of these assays to determine proglucagon expression relative to pGAPDH. Proglucagon Expression in Small Intestine The DCT values for pGAPDH and proglucagon transcript abundance (exps. 1 and 2) showed no significant interaction between intestinal location and treatment, allowing DCT values to be pooled and mean fold-change data to be presented across location and treatment. To illustrate the effect of small intestinal location on proglucagon expression, fold-change in proglucagon transcript abundance relative to the 50% of SI length location was determined. Expression of proglucagon, relative to 50% of SI length increased (P B0.05) by 1.2and 2.0-fold in exp. 1- and 1.5- and 2.0 fold in exp. 2 at 75 and 100% of SI length, respectively (Fig. 3). Expression level was significantly increased (P B0.05) between 75 and 100% of SI length in exp. 1, but was not significantly different in exp. 2. To illustrate the effect of treatment within experiment on proglucagon expression, fold-change in transcript abundance relative to CV pigs was calculated. In exp. 1, proglucagon expression was highest in LF pigs (1.6-fold CV) and different (P B0.05) from EC pigs (0.7-fold CV), which showed the lowest

proglucagon expression. Proglucagon expression was not different among LF, CV and GF pigs (Fig. 4). In exp. 2, relative proglucagon expression in LF pigs was 2.0-fold CV and significantly (P B0.05) higher than in EC and CV pigs similar to exp. 1. The SE contaminated group in exp. 2 showed a marked fourfold increase (P B0.05) in proglucagon expression relative to CV. Western Blot Assay for Active Caspase-3 Abundance In exp. 1, two separate protein bands were detected by immuno-staining. This double band has been previously reported (Mu et al. 2003) in Western blot assays detecting active caspase-3 protein (1719 kDa) in rats. In exps. 1 and 2, active caspase-3 protein abundance was 2.5 b

b

Exp. 1 Exp. 2

2 b Fold Scale

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1.5 a a

a

1

0.5

0 50%

75% 95% Small Intestine Location

Fig. 3. Mean fold difference in proglucagon expression relative to 50% of SI length for pigs maintained germ-free (GF), monoassociated with Lactobacillus fermentum (LF) or Escherichia coli (EC), Staphylococcus epidermidis (SE), or conventionalized with a slurry of adult porcine feces (CV). Bars within experiment not sharing a common letter are significantly different (PB0.05) and statistical differences are based on statistical analysis of DCT values.

SIGGERS ET AL. * PORCINE PROGLUCAGON AND MICROBIAL EFFECTS ON EXPRESSION

5

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Fold Scale

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EC

CV

Fig. 4. Mean fold difference in SI proglucagon expression for all treatment groups in exps. 1 and 2. In exp. 1, pigs were maintained germ-free (GF), monoassociated with Lactobacillus fermentum (LF) or Escherichia coli (EC), or conventionalized with adult porcine feces (CV). In exp. 2, the GF group was monoassociated with Staphylococcus epidermidis (SE). Average mean fold difference for all treatments was made relative to CV (CV 1.0). Bars within experiment not sharing a common letter are significantly different (PB0.05) and statistical differences are based on statistical analysis of ^CT values.

below detection levels for GF, SE, and LF pigs at 75% of SI length (Fig. 5). In exp. 1, caspase-3 protein was only detectable in CV pigs at 75% of SI. In exp. 2, caspase-3 protein showed strong immunostaining in CV pigs and faint immunostaining in EC pigs at 75% of SI length. DISCUSSION For more than 20 yr, proglucagon has been a major focus in human and animal gastrointestinal research. Proglucagon is a prohormone that is transcribed,

Fig. 5. Western blot assay for active caspase-3 (17 kDa) at 75% of small intestine length for all treatment groups in exps. 1 and 2. In exp. 1, pigs were maintained germ-free (GF), monoassociated with Lactobacillus fermentum (LF) or Escherichia coli (EC), or conventionalized with adult porcine feces (CV). In exp. 2, the GF group was monoassociated with Staphylococcus epidermidis (SE). Lane 1: SDS-PAGE molecular weight markers from largest to smallest (kDa): 103, 76, 49, 33.2, 28, 19.9; lane 25:GF, LF, EC, CV, respectively (exp. 1); lane 69: SE, LF, EC, CV, respectively (exp. 2); lane 10: human active caspase-3 (17 kDa) recombinant protein. Samples were pooled among animals within location for each treatment.

translated and differentially cleaved in the intestinal enteroendocrine L cells and the pancreatic a cells (Damholt et al. 1999). Many of the cleaved peptides from proglucagon have important biological activities in vivo. In particular, GLP-2 has emerged as a 33 amino acid peptide (proglucagon 126158) capable of increasing epithelial proliferation, decreasing epithelial apoptosis, and increasing nutrient absorption (Burrin et al. 2000). To date, in vivo experiments using pig models have used exogenous human GLP-2 analogues as a substitute for porcine GLP-2, perhaps because the two sequences are believed to be fairly conserved. Surprisingly, this is the first report of the nucleotide sequence of porcine proglucagon considering the pig has been used extensively to study GLP-2 physiology. Porcine proglucagon is highly similar among mammalian species with the exception of a short region at the carboxy-terminus. The nucleotide sequence of porcine proglucagon encodes amino acids serine and leucine at positions 159 and 160, respectively, versus arginine and lysine, respectively, in human, bovine and guinea pig. The predicted amino acid substitutions observed here in the pig are significant in that arginine and lysine are basic amino acids that are cleaved by a carboxypeptidase-B enzyme to yield GLP-2 (133) as in other mammals (Buhl et al. 1988). Serine and leucine are not susceptible to carboxypeptidase-B enzymatic cleavage suggesting that GLP-2 may be 35 amino acids in length in the pig. This amino acid substitution predicted by the nucleotide sequence of porcine proglucagon contrasts previous research by Buhl et al. (1988) and Ørskov et al. (1989). These studies employed GLP-2 peptide purification from pig intestinal tissue and epitope mapping to conclude that the carboxy-terminus of porcine GLP-2 was similar to human, bovine, and guinea pig GLP-2. Since epitope

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436 CANADIAN JOURNAL OF ANIMAL SCIENCE

mapping was conducted using antibodies raised against human carboxy-terminal peptides of proglucagon, the serine and leucine residues at 159 and 160 reported here may not have been detected. If the mature GLP-2 peptide is confirmed to be 35 versus 33 amino acids, then the biological activity of porcine GLP-2 (135) should be investigated. In the current study, we examined small intestinal physiology and proglucagon gene expression in isolatorreared conventionalized pigs, germ-free pigs and pigs monoassociated with a representative Gram-positive (L. fermentum) or Gram-negative (E. coli) commensal organism. In the second of two experiments, germ-free pigs were colonized with S. epidermidis, an environmental/skin contaminant. Cecal colonization level of monoassociated pigs was similar in both experiments, however, E. coli counts were 102 cfu g1 higher than L. fermentum. Cecal colonization rate of S. epidermidis was intermediate between E. coli and L. fermentum. Colonization rates were not determined for the SI, however, variation in colonization level should not be discounted as a possible factor contributing to our observations. Apoptosis is a highly regulated biological process, whereby damaged cells are destroyed and removed in a manner avoiding an inflammatory response in the host. Previously, apoptotic activity in the intestine has been measured by immunoblotting, DNA fragmentation, or radio-immunoassay techniques (Gupta 2003). Caspase-3 is an enzyme commonly measured to determine apoptotic activity (Janicke et al. 1998; Stadelmann and Lassmann 2000). Caspase-3 is synthesized as a proenzyme (32 kDA) that is processed in cells initiating apoptosis. The processed form of caspase-3 consists of two active subunits (17 and 12 kDa), which combine to form an active enzyme. Increased cellular abundance of the active subunits of caspase-3 is associated with increased apoptosis (Kothakota et al. 1997; Thornberry and Lazebnik 1998). In the current study, active caspase-3 subunit abundance in the distal SI was highest in CV pigs, intermediate in EC pigs (detectable in exp. 2 only), and below detection level in GF and monoassociated pigs. The differences in active caspase-3 abundance were consistent with changes in villus height and proteins involved in enterocyte turnover observed in the companion studies (Shirkey et al. 2006; Danielsen et al. 2007). Extremely long villi were observed in germ-free and lactobacilli treatment groups associated with undetectable levels of active caspase-3 and presumably low apoptotic rate. Escherichia coli and conventional pigs demonstrated villi of relatively intermediate and short length, respectively, consistent with high levels of active caspase-3 and increased apoptotic rate. These observations suggest that intestinal bacteria markedly influence epithelial cell replacement rate in the distal SI. Recently, scientists have discovered that microbes inhabiting the gastrointestinal tract can directly impact gastrointestinal health and function. More specifically,

these studies show that different bacteria can differentially affect gene expression within the host (Hooper et al. 2001; Kelly et al. 2004). Based on such findings, we used a gnotobiotic model to examine the effect of bacterial colonization on the expression of proglucagon, the GLP-2 prohormone and important regulator of intestinal function through modulation of epithelial cell replacement rate and nutrient absorption (Brubaker et al. 1997; Drucker 1999; Burrin et al. 2003). Proglucagon mRNA abundance was measured at different locations along the mid to distal region of the SI for all treatment groups. Although increased mRNA expression generally equates to increased protein abundance, occasionally gene transcription occurs in the absence of subsequent protein translation (Szymanski et al. 2003). Furthermore, since proglucagon yields many different active peptides, proglucagon mRNA abundance can not be directly equated to an increase in GLP2 activity. In both experiments, proglucagon expression increased from the mid to distal region of the SI for all treatment groups. Our observations reflect previous studies indicating that proglucagon expression increases distally along the SI as a result of the increasing proximal-to-distal gradient of enteroendocrine L cells from the jejunum to ileum (Eissele et al. 1992; Hoyt et al. 1996). Comparison of monoassociated pigs suggests that proglucagon expression is differentially responsive to the different bacterial species colonizing the digestive tract. Lactobacillus fermentum and S. epidermidis appeared to stimulate proglucagon expression, while the E. coli tended to be inhibitory. Among the monoassociated pigs, increased expression of proglucagon in LF and SE pigs was associated with longer villi compared with EC pigs, which demonstrated moderately shorter villi and detectable active caspase-3 in exp. 2. Escherichia coli pigs also had deeper crypts, which is consistent with increased epithelial cell proliferation. Generally, lactobacilli are acknowledged as a species of bacteria capable of beneficially impacting intestinal function (Kaur et al. 2002; Hebuterne 2003; Mack et al. 2003), while E. coli are considered more detrimental. Variation in proglucagon expression observed here may contribute to the beneficial or detrimental effects of these bacteria on the host. Although there is little evidence of the beneficial or detrimental effect of staphylococci bacteria on intestinal health and function, this contaminating organism was a potent stimulator of proglucagon expression. Further investigation will be required to determine the mechanism by which S. epidermidis and potentially other staphylococci stimulate proglucagon expression. Since short-chain fatty acids have been shown to stimulate proglucagon expression in the rat (Tappenden et al. 2002), the amount and profile of short-chain fatty acids produced by intestinal bacterial fermentation may be one mechanism contributing to the variation in proglucagon expression observed among

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SIGGERS ET AL. * PORCINE PROGLUCAGON AND MICROBIAL EFFECTS ON EXPRESSION

treatment groups. Clearly, further elucidation of the pathways involved could lead to new approaches to improve intestinal health and function. In contrast to the monoassociated groups, comparison of GF and CV pigs suggests that factors, independent of proglucagon expression, resulting from a complex microbiota regulate SI epithelial kinetics and morphology. At the extremes, proglucagon expression was similar between GF and CV pigs, whereas active caspase-3 was not detectable in GF versus easily detectable in CV, villi were two- to threefold longer in GF versus CV and crypts were significantly shorter in GF versus CV. Active caspase-3 abundance and SI morphology suggest a rapid epithelial cell turnover associated with a conventional microbiota, independent of the level of proglucagon expression. Obviously, other host factors could mediate epithelial turnover rate in response to bacterial colonization of SI including proinflammatory cytokines (Butzner and Gall 1988; Dignass and Podolsky 1993; Shirkey et al. 2006) and numerous other intestinal growth factors (Alison and Sarraf 1994). In addition, bacterial colonization may have direct effects on epithelial cell replacement rate through synthesis of toxins and production of toxic catabolites such as ammonia, factors known to increase apoptosis (Phillips et al. 2000; Keenan and Saini 2002). Thus, SI morphology and epithelial replacement rate are the net result of complex regulatory networks typical of a complex intestinal microbiota. We conclude that porcine GLP-2 may be 35 amino acids in length, based on the prediction of serine and leucine at positions 159 and 160 of porcine proglucagon. We confirm increased proglucagon expression from mid to distal SI in the pig and find that the differential effect of bacteria on small intestinal morphology may be mediated partially through differential regulation of proglucagon gene expression. The results support our previous studies demonstrating that differences in bacterial colonization during the neonatal period may be an approach to improving intestinal health and performance in the neonate.

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