Eric A. Wong

BOARD-INVITED REVIEW: Peptide absorption and utilization: Implications for animal nutrition and health

Over the last 50 yr, the study of intestinal peptide transport has rapidly evolved into a field with exciting nutritional and biomedical applications. In this review, we describe from a historical and current perspective intestinal peptide transport, the importance of peptides to whole-body nutrition, and the cloning and characterization of the intestinal peptide transporter, PepT1. We focus on the nutritional significance of peptide transport and relate these findings to livestock and poultry. Amino acids are transported into the enterocyte as free AA by a variety of AA transporters that vary in substrate specificity or as di- and tripeptides by the peptide transporter, PepT1. Expression of PepT1 is largely restricted to the small intestine in most species; however, in ruminants, peptide transport and activity is observed in the rumen and omasum. The extent to which peptides are absorbed and utilized is still unclear. In ruminants, peptides make a contribution to the portal-drained visceral flux of total AA and are detected in circulating plasma. Peptides can be utilized by the mammary gland for milk protein synthesis and by a variety of other tissues. We discuss the factors known to regulate expression of PepT1 including development, diet, hormones, diurnal rhythm, and disease. Expression of PepT1 is detected during embryological stages in both birds and mammals and increases with age, a strategic event that allows for the immediate uptake of nutrients after hatch or birth. Both increasing levels of protein in the diet and dietary protein deficiencies are found to upregulate the peptide transporter. We also include in this review a discussion of the use of dietary peptides and potential alternate routes of nutrient delivery to the cell. Our goal is to impart to the reader the nutritional implications of peptide transport and dietary peptides and share discoveries that shed light on various biological processes, including rapid establishment of intestinal function in early neonates and maintenance of intestinal function during fasting, starvation, and disease states.

Cloning of a turkey prolactin cDNA: expression of prolactin mRNA throughout the reproductive cycle of the domestic turkey (Meleagris gallopavo).

A cDNA-encoding turkey prolactin (PRL) has been isolated from a turkey pituitary library. The 953-base pair cDNA clone contains a 229-amino acid open reading frame which consists of a 30-amino acid signal peptide followed by a 199-amino acid mature PRL. The deduced amino acid sequence of turkey PRL shows greater than 90% homology to chicken PRL and 54-78% homology to other mammalian prolactins. A mRNA of 1100 nucleotides was detected in total RNA extracted from turkey pituitaries. Levels of PRL mRNA increased approximately 10-, 20-, and 100-fold in photostimulated, laying, and incubating hens, respectively, relative to that found in nonphotostimulated hens. The corresponding increases in plasma PRL levels were 2-, 5.5-, and 50-fold and in pituitary PRL content were 2-, 4-, and 13.4-fold, respectively. The transition from incubation to the photorefractory phase resulted in a 10-fold reduction in PRL mRNA, a 3.7-fold decrease in pituitary PRL, and a dramatic 50-fold decrease in plasma PRL. The changes in the abundance of pituitary PRL mRNA appear to be related to the changes in PRL-releasing activity observed at each of the reproductive stages. This study provides the first characterization of pituitary PRL mRNA and its comparison with plasma and pituitary PRL levels during the avian reproductive cycle.

Expression of ovine insulin-like growth factor-1 (IGF-1) stimulates alveolar bud development in mammary glands of transgenic mice

To determine whether murine mammary growth is modulated by local insulin-like growth factor-1 (IGF-1) production, expression of recombinant IGF-1 was directed to the mammary glands of transgenic mice using an ovine prepro IGF-1 cDNA under control of the mouse mammary tumor virus-long terminal repeat (MMTV-LTR) promoter. Bioactivity of recombinant IGF-1 in transgenic mouse milk extracts was demonstrated by a concentration-dependent increase in [3H]thymidine incorporation in clonal bovine mammary epithelial cells (MAC-T) compared with control mouse milk extracts; moreover, addition of excess recombinant human insulin-like growth factor binding protein-3 (rhIGFBP-3) abolished the increase in [3H]thymidine incorporation attributed to recombinant IGF-1 in transgenic mouse milk. Recombinant IGF-1 was produced in mammary tissue of virgin and pregnant transgenic mice, and secreted into milk of lactating mice. However, recombinant IGF-1 was not detected in serum from transgenic mice; and ligand blot analysis of serum insulin-like growth factor binding proteins (IGFBPs) indicated no differences owing to transgene presence. In peripubertal virgin mice at 49 d of age, the frequency of appearance of mammary alveolar buds was significantly higher in MMTV-IGF-1 than in CD-1 mice, and was unaffected by ovariectomy or estradiol treatment. In conclusion, mammary synthesis of recombinant IGF-1 enhances the rate of development of alveolar buds in mammary glands of virgin transgenic mice.

Gene Expression of Nutrient Transporters in the Small Intestine of Chickens from Lines Divergently Selected for High or Low Juvenile Body Weight

Nutrient transporters in the small intestine are responsible for dietary nutrient assimilation; therefore, the expression of these transporters can influence overall nutrient status as well as the growth and development of the animal. This study examined correlated responses to selection in the developmental gene expression of PepT1, EAAT3, SGLT1, and GLUT5 in the small intestine of chickens from lines divergently selected for 48 generations for high (HH) or low (LL) 56-d BW and their reciprocal crosses (HL and LH). Duodenum, jejunum, and ileum were collected from male and female chicks on embryonic d 20, day of hatch with no access to feed, and d 3, 7, and 14 posthatch. Total RNA was extracted, and nutrient transporter expression was assayed by real-time PCR using the relative quantification method. In comparing male and female HH and LL chicks, there was a mating combination x age x sex interaction for PepT1 expression (P < 0.001), a main effect of sex for EAAT3 (P < 0.05) and SGLT1 (P < 0.001) expression, and an age x sex interaction for SGLT1 expression (P < 0.001). These results demonstrate a sexual dimorphism in the capacity to absorb nutrients from the intestine, which has implications for the poultry industry with regard to diet formulations for straight-run and sex-separate grow-out operations. Results from comparing male LL, LH, HL, and HH chicks indicate that selection for high or low juvenile BW may have influenced the gene expression profiles 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.

Vasoactive intestinal peptide stimulates prolactin mRNA expression in turkey pituitary cells: effects of dopaminergic drugs.

Abstract It is well documented that vasoactive intestinal peptide (VIP) is a prolactin (PRL)-releasing factor and that dopamine (DA) is an inhibitory neurotransmitter in avian species. However, the roles of VIP and DA in the regulation of PRL gene expression are unclear. In this study, primary anterior pituitary cells cultured from laying turkeys were utilized to investigate the influence of VIP and dopaminergic D1 and D2 receptors on PRL secretion, PRL mRNA, and PRL synthesis. Incubation of pituitary cells with VIP increased PRL secretion up to 3.5-fold within 3 hr. Prolactin mRNA was undetectable during the first 2 hr of pituitary cell treatment; thereafter, the PRL mRNA content response to VIP increased within 24-48 h (P < 0.05). Total PRL content (media + cellular) increased over time in the presence of VIP. The response of cells incubated in the presence of a dopaminergic D1 receptor agonist (SKF38393) was variable and inconclusive. However, cells incubated with a dopaminergic D2 receptor agonist (quin-pirole) inhibited VIP-induced PRL secretion (P < 0.05) and PRL mRNA levels (P < 0.05) in a dose-related fashion without effect on the basal levels of PRL release and PRL mRNA. These observations suggest that VIP, in addition to acting as a PRL-releasing peptide, also plays a role in the regulation of PRL gene expression. Moreover, the results of this study also indicate that a drug that can selectively stimulate dopamine D2 receptors can also regulate PRL secretion and PRL mRNA in turkey pituitary cells in culture. [P.S.E.B.M. 1996, Vol 212]

Towards a species-selective acetylcholinesterase inhibitor to control the mosquito vector of malaria, Anopheles gambiae

Anopheles gambiae is the major mosquito vector of malaria in sub-Saharan Africa. At present, insecticide-treated nets (ITNs) impregnated with pyrethroid insecticides are widely used in malaria-endemic regions to reduce infection; however the emergence of pyrethroid-resistant mosquitoes has significantly reduced the effectiveness of the pyrethroid ITNs. An acetylcholinesterase (AChE) inhibitor that is potent for An. gambiae but weakly potent for the human enzyme could potentially be safely deployed on a new class of ITNs. In this paper we provide a preliminary pharmacological characterization of An. gambiae AChE, discuss structural features of An. gambiae and human AChE that could lead to selective inhibition, and describe compounds with 130-fold selectivity for inhibition of An. gambiae AChE relative to human AChE.

IGF-I-Induced IGFBP-3 potentiates the mitogenic actions of IGF-I in mammary epithelial MD-IGF-I cells

Limited information is available concerning the molecular and cellular mechanisms that regulate expression of insulin-like growth factor-I (IGF-I) binding proteins (IGFBPs) in bovine mammary epithelial cells. Here, we report on the autocrine mechanisms of action of IGF-I and hormonal regulation of expression of IGFBPs in bovine mammary epithelial MD-IGF-I cells which express recombinant IGF-I under the control of the glucocorticoid-inducible mouse mammary tumor virus-long terminal repeat (MMTV-LTR). Levels of IGFBP-3 mRNA and secretion of IGFBP-3 by MD-IGF-I cells were stimulated by IGF-I, insulin (INS), and IGF-I analogs but not prolactin (PRL). Conversely, parental MAC-T cells expressed little IGF-I and secreted primarily IGFBP-2 (29-32 kDa) in response to stimulation with INS, dexamethasone (DEX), or IGF-I analogs. Secretion of recombinant IGF-I caused a 26.5-fold increase in secretion of IGFBP-3, as measured by densitometric analysis of ligand blots, which was associated with a 1.7-fold increase in total DNA. Conditioned media (CM) from MD-IGF-I cells induced with DEX stimulated a 2.8-fold increase in [3H]thymidine incorporation into DNA of parental MAC-T cells, compared with uninduced cells. Moreover, inclusion of exogenous IGF-I with CM from MD-IGF-I cells triggered an additional 3.0-fold increase in label incorporation, but only a 1.6-fold increase in the presence of IGFBP-2-containing media conditions by MAC-T cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Gene expression of nutrient transporters and digestive enzymes in the yolk sac membrane and small intestine of the developing embryonic chick.

Chick embryos derive nutrients from the yolk during incubation and transition to intestinal absorption of nutrients posthatch. The uptake of nutrients is mediated by a variety of membrane-bound transporter proteins. The objective of this study was to determine the expression profiles of nutrient transporters and digestive enzymes during incubation in the yolk sac membrane (YSM) and embryonic intestine of egg-laying (Leghorn) and meat-producing (Cobb) chickens derived from 22 to 30 wk (young) and 45 to 50 wk (old) breeder flocks. Transporters examined included the peptide transporter PepT1, the glutamate/aspartate (EAAT3), cationic (CAT-1) and neutral (B0AT) amino acid transporters, and the fructose (GLUT5) and glucose (SGLT1) transporters. Digestive enzymes included aminopeptidase N (APN) and sucrase-isomaltase (SI). Expression of these genes was assessed by real-time PCR using the absolute quantification method in YSM at embryonic day (E) 11, 13, 15, 17, 19, 20, and 21 and intestine at E15, 17, 19, 20, and 21. The PepT1 and APN gene expression in the YSM increased until E15 and then decreased until E21, whereas expression in the intestine increased from E15 to E21. The B0AT showed a similar pattern, with greatest expression in the YSM occurring at E17/E19. The CAT1 and GLUT5 genes showed decreased expression in the YSM and increased expression in the intestine until E17/E19 and then a decrease until E21. Expression of SGLT1 and EAAT3 showed increased gene expression over time in both the intestine and YSM. Expression of SI showed little to no gene expression in the YSM, whereas the intestine exhibited consistently high levels of gene expression. In YSM and intestine, SI expression was greater in Leghorn than Cobb, whereas CAT1 and GLUT5 expression was greater in Cobb than Leghorn. Expression of the APN, CAT1, and SI genes was greater in embryos from young flocks than old flocks in YSM and intestine. These results demonstrate that the YSM expresses many of the digestive enzymes and nutrient transporters typically associated with the intestine and that these genes show tissue- and development-specific patterns of expression.

The turkey prolactin-encoding gene and its regulatory region ☆

Overlapping prolactin (Prl) lambda clones were isolated from a turkey genomic library. The 6.7-kb turkey Prl gene consists of five exons. Major transcription start points were located by primer extension 51-53 nucleotides upstream from the Met start codon. No estrogen response element (ERE) was found, but two regions similar to mammalian Pit-1/GHF-1-binding sites were identified by computer analysis. This suggests that transcription of the turkey Prl gene may be regulated by Pit-1/GHF-1, and not by the estrogen receptor.

Expression profiling of the solute carrier gene family in chicken intestine from the late embryonic to early post‐hatch stages

Intestinal development during late embryogenesis and early post-hatch has a long-term influence on digestive and absorptive capacity in chickens. The objective of this research was to obtain a global view of intestinal solute carrier (SLC) gene family member expression from late embryogenesis until 2 weeks post-hatch with a focus on SLC genes involved in uptake of sugars and amino acids. Small intestine samples from male chicks were collected on embryonic days 18 (E18) and 20 (E20), day of hatch and days 1, 3, 7 and 14 post-hatch. The expression profiles of 162 SLC genes belonging to 41 SLC families were determined using Affymetrix chicken genome microarrays. The majority of SLC genes showed little or no difference in level of expression during E18-D14. A number of well-known intestinal transporters were upregulated between E18 and D14 including the amino acid transporters rBAT, y(+)LAT-2 and EAAT3, the peptide transporter PepT1 and the sugar transporters SGLT1, GLUT2 and GLUT5. The amino acid transporters CAT-1 and CAT-2 were downregulated. In addition, several glucose and amino acid transporters that are novel to our understanding of nutrient absorption in the chicken intestine were discovered through the arrays (SGLT6, SNAT1, SNAT2 and AST). These results represent a comprehensive characterization of the expression profiles of the SLC family of genes at different stages of development in the chicken intestine and lay the ground work for future nutritional studies.