Browsing by Author "Miller, Carin R."

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    Developmental Gene Expression in the Small Intestine of Chickens from Lines Divergently Selected for High or Low Juvenile Body Weight
    Miller, Carin R. (Virginia Tech, 2007-09-10)
    Nutrient transporters in the small intestine are responsible for dietary nutrient assimilation and therefore the expression of these transporters can influence the overall nutrient status as well as the growth and development of the animal. This thesis examined correlated responses to selection in the developmental gene expression of the peptide transporter PepT1, the glutamate/aspartate transporter EAAT3, the sodium-dependent glucose transporter SGLT1, and the fructose transporter GLUT5 in the small intestine of chickens from lines divergently selected for high (HH) or low (LL) eight-week body weight and their reciprocal crosses, (HL and LH). Chicks were weighed and killed on embryonic day 20 (E20), day of hatch (DOH with no access to feed), and days 3 (D3), 7(D7), and 14 (D14) post hatch. Duodenum, jejunum, ileum and liver were collected. DNA extracted from liver was used to sex birds by PCR. RNA was extracted from the intestinal segments of four males and four females from each mating combination (MC) and time point except E20 HL males (n = 3) and D7 LL females (n = 2). Expression of nutrient transporters was assayed by real-time PCR using the relative quantification method. In comparing HH and LL males and females there was a line by segment interaction in PepT1 gene expression, with no segment difference in HH and greatest expression in the ileum of the LL (P < 0.05). There was also a MC by age by sex interaction for PepT1 gene expression (P < 0.0001) with peak gene expression occurring on DOH for LL females, on D7 for HH females, on D7 for LL males and D14 for HH males. Overall, females had greater EAAT3 expression (P < 0.03). Gene expression of EAAT3 was greatest in the ileum, intermediate in the jejunum, and least in the duodenum (P < 0.0007). There was an age by segment interaction for EAAT3 expression (P = 0.0002) and a MC by segment interaction (P < 0.02), with LL having greater expression than HH in the ileum. Females had greater SGLT1 expression than males (P < 0.0001). There was a sex by age interaction for the expression of SGLT1 (P < 0.0001). Females induced SGLT1 expression on DOH and maintained this level through D14, while males gradually increased expression through D7 and decreased expression by D14. These results indicate that expression of PepT1, EAAT3, SGLT1 are differentially expressed in male and female chickens regardless of selection for high or low juvenile body weight. These results also show a sexual dimorphism in the capacity to absorb peptides, anionic amino acids, and glucose from the intestine, which has implications for the poultry industry with regard to diet formulations for straight-run and sex-separate grow-out operations. In comparing male HH, HL, LH, and LL chicks, overall LL had the greatest level of expression (P <0.06), HH had the least level of expression (P < 0.006) and HL and LH had intermediate levels of expression (P < 0.06). Greatest PepT1 gene was expression in the ileum (P < 0.0003) and there was a MC by segment interaction with expression increasing from duodenum to ileum in LL, but there was no segment difference in any other MC (P < 0.08). Within each intestinal segment there was a MC difference (P < 0.02). There was an effect of sire for PepT1 expression, with progeny from low weight selected sires (LWS) having greater expression than progeny from high weight selected (HWS) sires (P = 0.0008). There was no difference between intestinal segments in progeny from HWS sires, however, greatest PepT1 gene expression was seen in the ileum of progeny from LWS sires (P < 0.0001). Overall, expression of EAAT3 was greatest in the ileum, intermediate in the jejunum and least in the ileum (P < 0.0001) and there was a segment by age interaction for EAAT3 expression (P < 0.0001). In all MCs except HH, EAAT3 gene expression increased from duodenum to ileum (P < 0.08). Within the ileum, the LL had greatest EAAT3 gene expression, LH and HL had intermediate gene expression, and HH had least expression (P < 0.08). Expression of SGLT1 gradually increased through D7 and decreased by D14 (P < 0.0001) and overall, was greatest in the distal small intestine (P < 0.0001). There was a MC by segment interaction, with SGLT1 gene expression being greatest in the distal small intestine in LL, LH, and HL, but greatest in the jejunum of HH (P < 0.04). Within the ileum, LL had greater SGLT1 gene expression than HH (P < 0.06). Overall, greatest GLUT5 expression was in the distal small intestine (P < 0.0001) and there was a MC by segment interaction, with expression being greatest in the distal small intestine in LL and HL (P < 0.02), greatest in the ileum of LH (P < 0.08), and greatest in the jejunum of HH (P < 0.09). Within the ileum there was a MC difference (P < 0.07). These results indicate that selection for high or low juvenile body weight may have influenced the gene expression pattern of these nutrient transporters in the small intestine, which may contribute to the overall differences in the growth and development of these lines of chickens.
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    Regulatory Balance Between the Peptide Trasporter, Pept1, and Amino Acid Transporter Gene Expression in the Enterocyte
    Miller, Carin R. (Virginia Tech, 2012-04-04)
    Amino acids are assimilated by membrane-associated transporters into and out of enterocytes either in their free form or in the form of peptides. The peptide transporter, PepT1, is thought to be the major facilitator of peptide transport in the enterocyte. It is unknown if the peptide transporters and free amino acid transporters operate in a compensatory fashion to regulate the amino acid balance within the enterocyte. Therefore, the objective was to examine the regulatory balance between PepT1 and other peptide and free amino acid transporters in enterocytes. The Mouse Small Intestinal Epithelial (MSIE) cells are conditionally immortalized. It was found that MSIE cells express BoAT1, CAT1, CAT2, LAT1, y+LAT1, and y+LAT2, but not PepT1, EAAT3, Bo,+AT, or LAT2, making this model similar to the basolateral membrane of enterocytes. Growing MSIE cells at high temperatures did not affect the nutrient transporter gene expression profile of these cells. Thus, the human colon carcinoma (Caco-2) cell line was used as a small intestinal in vitro model for this study. These cells express PepT1, HPT1, PTR3 EAAT1, EAAT3, rBAT, Bo,+AT CAT1, LAT1, y+LAT1, y+LAT2, ABCC3, ABCC4, which increased from D0 to D21 post confluency, indicating cell maturation. In Caco-2 cells, PepT1 gene silencing was induced in Caco-2 cells. Despite an reduction of PepT1 gene (82%, P < 0.05) protein (96%), no significant difference in any peptide (HPT1, PTR3, ABCC3, ABCC4) or free amino acid transporters (EAAT1, EAAT3, rBAT, Bo,+AT, BoAT1, CAT1, CAT2, LAT1, LAT2, y+LAT1, y+LAT2) between Caco-2 cells treated with PepT1 siRNA and Caco-2 cells treated with Control siRNA was observed. These results suggest no compensation at the gene expression level of these transporters in response to a reduction of PepT1. To account for the limitations of an in vitro and PepT1 kockout mouse model, transgenic chicken models were pursued. Potential cPepT1 overexpressing, cPepT1 shRNA or control shRNA expressing G0 chickens were generated by embryo injection of pseudolentiviral particles followed by ex ovo egg culture. Overall, 9 potential G0 cPepT1 overexpressing chickens, 15 potential G0 cPepT1 shRNA expressing chickens, and 4 potential G0 control shRNA expressing chickens were generated.