Peptide Absorption and Utilization: Implications for Animal Nutrition and Health E. R. Gilbert, E. A. Wong and K. E. Webb, Jr. J Anim Sci published online Apr 25, 2008; The online version of this article, along with updated information and services, is located on the World Wide Web at: http://jas.fass.org www.asas.org Downloaded from jas.fass.org by on April 3, 2010. Page 1 of 74 Journal of Animal Science Review: Peptide Absorption and Utilization 1 Peptide Absorption and Utilization: Implications for Animal Nutrition and Health 2 3 E. R. Gilbert*, E. A. Wong*, and K. E. Webb, Jr.* 4 * 5 Department of Animal and Poultry Sciences, 6 7 Virginia Polytechnic Institute and State University, 8 9 Blacksburg, VA 24061-0306 10 11 Corresponding author: 12 13 K. E. Webb, Jr. 14 3470 Litton Reaves Hall 15 Blacksburg, VA 24061-0306 16 Phone: (540) 231-9157 17 Fax: (540) 231-3010 18 Email: email@example.com 1 First on from jas.fass.org by as doi:10.2527/jas.2007-0826 Published OnlineDownloaded April 25, 2008 on April 3, 2010. Journal of Animal Science Page 2 of 74 19 20 ABSTRACT: Over the last 50 years, the study of intestinal peptide transport has 21 rapidly evolved into a field with exciting nutritional and biomedical applications. In this 22 review we describe from a historical and current perspective intestinal peptide transport, 23 the importance of peptides to whole-body nutrition, and the cloning and characterization 24 of the intestinal peptide transporter, PepT1. We focus on the nutritional significance of 25 peptide transport and relate these findings to livestock and poultry. Amino acids are 26 transported into the enterocyte as free amino acids by a variety of amino acid transporters 27 that vary in substrate specificity or as di- and tripeptides by the peptide transporter, 28 PepT1. Expression of PepT1 is largely restricted to the small intestine in most species; 29 however, in ruminants, peptide transport and activity is observed in the rumen and 30 omasum. The extent to which peptides are absorbed and utilized is still unclear. In 31 ruminants, peptides make a contribution to the portal-drained visceral flux of total amino 32 acids and are detected in circulating plasma. Peptides can be utilized by the mammary 33 gland for milk protein synthesis and by a variety of other tissues. We discuss the factors 34 known to regulate expression of PepT1 including development, diet, hormones, diurnal 35 rhythm, and disease. PepT1 is detected during embryological stages in both birds and 36 mammals and increases with age, a strategic event that allows for the immediate uptake 37 of nutrients after hatch or birth. Both increasing levels of protein in the diet and dietary 38 protein deficiencies are found to upregulate the peptide transporter. We also include in 39 this review a discussion of the use of dietary peptides and potential alternate routes of 40 nutrient delivery to the cell. Our goal is to impart to the reader the nutritional implications 41 of peptide transport and dietary peptides and share discoveries that shed light on various 42 biological processes, including rapid establishment of intestinal function in early 2 Downloaded from jas.fass.org by on April 3, 2010. Page 3 of 74 Journal of Animal Science 43 neonates and maintenance of intestinal function during fasting, starvation, and disease 44 states. 45 Key words: AA absorption, dietary regulation, intestine, PepT1, peptides, transporter 46 INTRODUCTION 47 “If we continue to look only for free amino acids, we shall find only free amino 48 acids: peptides cannot be expected to declare their presence”. This was Matthews’ (1991) 49 eloquent way of stating that the contribution of peptides to total AA absorption and 50 utilization had been ignored for far too long. Long before the cloning and characterization 51 of the intestinal peptide transporter, PepT1 (Fei et al., 1994), a carrier-mediated 52 mechanism was shown to exist for the uptake of small peptides across the brushborder 53 membrane of the enterocyte. Although the first evidence for peptide transport was 54 provided in the 1950’s (Newey and Smyth, 1959, 1960), acceptance of AA absorption 55 across the gut wall in the form of peptides was slow to emerge, even as recently as the 56 late 1980’s (Webb, 1990). In addition to transport through PepT1, peptides may also be 57 absorbed through alternate routes including paracellular movement and by cell- 58 penetrating peptides (CPP) that are capable of moving cargo across the plasma membrane 59 (Figure 1). The purpose of this review is to describe, both from a historical and current 60 perspective, intestinal peptide transport, which has been characterized primarily in 61 laboratory species such as the rat and relate these findings to livestock and poultry. We 62 hope that, through this review, we can impart to the reader the nutritional importance of 63 peptide transport in agriculturally important animals and with this knowledge, the 64 potential to advance the field of nutrition and diet formulation. Unless it is otherwise 65 stated, the use of the term peptide will imply collectively di- and tripeptides. While 3 Downloaded from jas.fass.org by on April 3, 2010. Journal of Animal Science Page 4 of 74 66 excellent reviews on peptide transport and the intestinal peptide transporter, PepT1, have 67 been previously published (Webb, 1990; Webb et al., 1993; Leibach and Ganapathy, 68 1996; Daniel, 2004; Daniel and Kottra, 2004), this is the first to combine information 69 from classical nutrition studies with recent molecular and gene regulatory studies to 70 define a novel approach to how we view protein nutrition and gut physiology across a 71 wide range of species. 72 FATE OF LUMINAL PROTEIN DIGESTION PRODUCTS 73 The end-products of stomach digestion enter the small intestine where pancreatic 74 enzyme digestion (trypsin, chymotrypsin, elastase, etc.) begins the process of generating 75 absorbable end-products. Hydrolases are expressed on the brushborder membrane of 76 absorptive, epithelial cells serving to further digest luminal nutrients. The end products of 77 digestion are absorbed by these epithelial cells in the intestine through the action of 78 nutrient transporters located on the brushborder membrane. There are differences in the 79 nutrient requirements of cells throughout the body and a plethora of transporters are 80 present with varying structures and functions. Transport systems consist of proteins that 81 recognize, bind, and relocate a substrate or multiple substrates across a cell membrane. 82 Transporters have been characterized in endothelial cells and in the apical and basolateral 83 membranes of epithelial cells throughout the body. Amino acids can be transported 84 across the brushborder membrane of intestinal epithelial cells either in their free form by 85 a variety of different AA transporters that vary in substrate specificity or in the form of 86 di- and tripeptides by the broad-specificity peptide transporter, PepT1. Among the AA 87 transporters that have been well-characterized are transporters that are specific for basic 88 AA (rBAT and bo,+AT, CAT1 and 2, y+LAT1 and 2), neutral AA (BoAT, LAT1), and 4 Downloaded from jas.fass.org by on April 3, 2010. Page 5 of 74 Journal of Animal Science 89 anionic AA (EAAT3), to name a few, with most of these transporters exhibiting some 90 substrate overlap and various ion dependencies and mechanisms for movement of AA 91 across the cell membrane (Albritton et al., 1989; Kanai and Hediger, 1992; Segawa et al., 92 1997; Chairoungdua et al., 1999; Rajan et al., 2000; Broer et al., 2004). 93 PEPTIDE TRANSPORT VS FREE AA TRANSPORT 94 While transporters of free AA exhibit substrate specificity, PepT1 can potentially 95 transport all 400 di- and 8,000 tripeptides that result from combining the 20 different 96 dietary AA (Daniel, 2004). In terms of energetic efficiency, two or three AA can be 97 transported into the cell by PepT1 for the same expenditure of energy required to 98 transport a single free AA (Daniel, 2004). In addition, individuals suffering from 99 deficiencies in free AA transport were still able to assimilate essential AA, pointing to the 100 possibility that PepT1 transports enough dietary AA to compensate for a deficiency in 101 free AA transport (Adibi, 1997). 102 Transport of AA in the form of peptides was demonstrated to be a faster route of 103 uptake per unit time than their constituent AA in the free form (Adibi and Phillips, 1968; 104 Craft et al., 1968; Adibi, 1971; Cheng et al., 1971; Burston et al., 1972; Adibi, 1986). In 105 rats, AA in soy protein hydrolysate (Kodera et al., 2006) or egg white protein hydrolysate 106 (Hara et al., 1984) were absorbed faster into the portal blood after duodenal infusion than 107 those representing an AA mixture or intact protein with the same respective amino acid 108 composition. Similar results were observed using milk protein hydrolysate vs. a free AA 109 mixture in pigs (Rerat et al., 1988) and humans (Silk et al., 1980). Intestinal perfusion of 110 0, 50, 100, or 200 g/L solutions of intact soy protein or 0, 100, 200, 300, or 400 g/L 111 solutions of hydrolyzed soy protein caused a load-dependent slowing of intestinal transit 5 Downloaded from jas.fass.org by on April 3, 2010. Journal of Animal Science Page 6 of 74 112 in dogs (Zhao et al., 1997). Interestingly, the intact protein slowed transit more 113 effectively than the hydrolysate, supporting the notion that digestion is a rate-limiting 114 step in nutrient assimilation. Furthermore, a greater amount of AA were absorbed in the 115 proximal small intestine when protein was infused as a hydrolysate instead of in the intact 116 form, suggesting that AA as peptides were more readily available for absorption. 117 PEPTIDES AND PORTAL DRAINED VISCERA AA FLUX 118 The matter of intestinal AA flux is further complicated by the presence of 119 significant mucosal AA catabolism as dietary AA are the preferred fuel over glucose. 120 This may partially explain variability and discrepancies of past studies (Wu, 1998; Stoll 121 and Burrin, 2006). For example, 90% of dietary glutamate is sequestered by the portal- 122 drained viscera (PDV) in swine. Intestinal mucosal cells use AA for energy, protein 123 synthesis, nucleosides, polyamides, and maintenance of the intestinal immune system 124 (i.e, glutathione and mucin synthesis; Reeds et al., 2000). 125 Thus, although it is certain that AA derived from dietary protein are transported 126 into the enterocyte in the form of di- and tripeptides, there is still much to be learned 127 about the quantitative significance and origin of luminally-derived peptides and the 128 relative post-absorptive use and portal appearance of free AA vs. peptides. Determination 129 of amounts of free and peptide-bound AA in plasma involves protein removal followed 130 by amino acid analysis. A variety of plasma deproteinization methods are used, including 131 chemical precipitation with sulfosalicylic acid (Koeln et al., 1993) or methanol (Tagari et 132 al., 2004, 2008), physical removal using ultrafiltration (Seal and Parker, 1996), or a 133 combination of chemical precipitation followed by the ultrafiltration step (Han et al., 134 2001b; Tagari et al., 2004, 2008). The traditional method of AA analysis is separation by 6 Downloaded from jas.fass.org by on April 3, 2010. Page 7 of 74 Journal of Animal Science 135 ion-exchange chromatography (Koeln et al., 1993) in which samples are applied to a 136 column and eluted by buffers with increasing pH and/or ionic strength (White et al., 137 1986). Post-column derivatization with ninhydrin generates a colorimetric product that 138 can be measured. The Pico-Tag Waters method (Seal and Parker, 1996; Delgado-Elorduy 139 et al., 2002; Tagari et al., 2004, 2008) is a newer approach in which phenylisothiocyanate 140 (PITC) is used for pre-column derivatization of AA. Following derivatization, reversed- 141 phase gradient elution high-performance liquid chromatography separates the PITC 142 derivatives that are quantified based on UV absorbance. In simplest terms, peptide-bound 143 AA are calculated as the difference between total AA observed following hydrolysis of 144 protein-free preparations and free AA present prior to hydrolysis. Based on the different 145 combinations of methods for amino acid analysis, there are likely to be variations in 146 quantities of plasma peptides reported in the literature. 147 Little or no detection of absorbed intact peptides was reported in dogs (Levenson 148 et al., 1959) and rats (Wiggans and Johnson, 1959). Others have reported that up to 85 % 149 of AA entering the portal blood were in the form of small peptides in rats (Gardner, 150 1975), Holstein calves (Koeln et al., 1993), lactating Holstein cows (Tagari et al., 2004, 151 2008), Friesian steers (Seal and Parker, 1996), sheep (Remond et al., 2000), and yak cows 152 (Han et al., 2001a,b). In some of these studies, total appearance of free and peptide AA in 153 the portal vein was greater than the intake of dietary protein, suggesting contributions 154 from degradation products resulting from tissue protein turnover in the G. I. tract, spleen, 155 and pancreas (Han et al., 2001a). In studies where lower contributions of peptide-bound 156 AA to total portal amino acid flux were observed (32 %, Remond et al., 2000 and Han et 157 al., 2001b; up to 20 %, Tagari et al., 2004, 2008), the authors attributed the conflicting 7 Downloaded from jas.fass.org by on April 3, 2010. Journal of Animal Science Page 8 of 74 158 reports to differences in methodology. Refinements in analytical technique, including 159 chemical deproteinization of plasma followed by physical filtration, result in a more 160 accurate determination of peptide AA concentrations. The more recent reports indicating 161 a lower PDV flux of peptides may represent a more reasonable estimate of this value. 162 Although estimates of the proportion and absolute concentration of peptide AA entering 163 the portal circulation vary across and within species, the fact still remains that peptides 164 are detected in the blood and their concentration is influenced by diet suggesting their 165 importance as a nutritional resource to the liver and extra-hepatic tissues. 166 DiRienzo (1990) quantified the in-vivo flux of both free and peptide AA across 167 the mesenteric and nonmesenteric portions of the PDV in both calves and sheep and 168 made two important findings: 1) the mesenteric flux (drainage into mesenteric vein from 169 jejunum, ileum, cecum, colon and pancreas) of free and peptide AA was similar, and 2) 170 the flux of peptide AA across the nonmesenteric-drained viscera (from rumen, reticulum, 171 omasum, abomasum, duodenum and spleen) contributed the largest proportion of total 172 PDV AA flux in both calves and sheep. This study confirmed the contribution of peptides 173 to the portal appearance of AA and also demonstrated significant uptake of peptides from 174 the rumen and omasum. This was later confirmed by Matthews and Webb (1995), 175 McCollum and Webb (1998), and by Tagari et al. (2004, 2008). This observation has 176 important nutritional implications in ruminants where dietary supply of AA nitrogen and 177 microbial metabolism of dietary protein influences the nutrients that are made available 178 to the animal. 179 Matthews and Webb (1995) found uptake of two dipeptides and free methionine 180 across sheep ruminal and omasal epithelia to occur by a non-carrier mediated route of 8 Downloaded from jas.fass.org by on April 3, 2010. Page 9 of 74 Journal of Animal Science 181 uptake as demonstrated by a linear response in uptake with greater absorption in omasal 182 tissues as compared with ruminal tissues. Because serosal appearance of peptides was a 183 non-saturatable process, the data suggested that absorption occurred by a non carrier- 184 mediated process. However, in a later report characterizing a peptide transporter in 185 omasal epthelia (Matthews et al., 1996), the authors mentioned that attempts to identify 186 saturatable uptake of several dipeptides may have been confounded by the use of 187 substrate concentrations that were greater than mediated transport capacity. 188 FATE OF PLASMA PEPTIDES 189 Circulating peptides may not be hydrolyzed in plasma and there is negligible 190 clearance of peptides from plasma by erythrocytes (Lochs et al., 1990; Odoom et al., 191 1990). Peptides are likely transported into cells and hydrolyzed into free AA which are 192 then used for protein synthesis (Krzysik and Adibi, 1979) with the extent of peptide 193 utilization in cells influenced by dietary status (Webb, 1990; Webb et al., 1993). In calves 194 fed a diet of hay, corn, and soybean meal, circulating peptide AA were present at a 195 threefold higher concentration than free AA with a preferential removal of peptide AA 196 from plasma by tissues of the hind limbs (McCormick and Webb, 1982). When fed 197 purified diets containing either urea or soybean meal as the sole supplier of nitrogen, 198 peptide removal varied depending on the protein source with greatest removal by the hind 199 limbs in calves fed the soybean meal and negligible removal by the hind limbs in calves 200 fed the urea (Danilson et al., 1987). These results suggest that diet influences 201 concentrations of circulating peptides and availability to extra-hepatic tissues. That there 202 is tissue selectivity for peptide removal suggests that there may be tissue-specific ability 203 to utilize circulating plasma peptides. 9 Downloaded from jas.fass.org by on April 3, 2010. Journal of Animal Science Page 10 of 74 204 The hydrophobicity of peptides, which may also be related to dietary protein 205 source, may affect their absorption and subsequent utilization by tissues. Hydrophobic 206 peptides and peptides resistant to mucosal-hydrolysis were utilized or absorbed at a faster 207 rate than peptides that are hydrophilic and susceptible to hydrolysis in C2C12 myogenic 208 and MAC-T mammary cells (Pan et al., 1996). Burston and Matthews (1990); however, 209 did not find a correlation between hydrophobicity and transport rate of peptides in 210 hamster jejunum. Pan and Webb (1998) demonstrated that molecular structure influenced 211 availability of methionine-containing peptides as a methionine source in ovine skeletal 212 muscle cells. There were distinct differences in utilization of methionine-containing 213 dipeptides in three cell types suggesting that there were cell-specific differences in 214 transport and hydrolytic events. 215 PEPTIDES AS SOURCE OF AA FOR THE MAMMARY GLAND 216 One particular area of interest is how peptides may supply essential AA to 217 mammary tissue for milk protein synthesis, especially in animals used for milk 218 production. The literature contains multiple reports indicating that these AA requirements 219 may not be met based on estimates of free AA coming from the blood (Guinard and 220 Rulquin, 1994; Metcalf et al., 1996; Bequette et al., 1999). Wang et al. (1996) evaluated 221 peptide-bound methionine as a source for protein synthesis in mammary tissue explants 222 from lactating mice and found no difference in the ability of five of seven pairs of 223 dipeptides to stimulate [3H]-leucine incorporation into proteins, but the di- and tripeptides 224 promoted a greater incorporation than did free methionine. Although PepT1 mRNA was 225 not detected in mammary cells (Chen et al., 1999), aminopeptidase N mRNA was 226 detected in caprine mammary cells and found to be regulated by the postabsorptive form 10 Downloaded from jas.fass.org by on April 3, 2010. Page 11 of 74 Journal of Animal Science 227 of circulating AA (Mabjeesh et al., 2005), thus suggesting a mechanism for utilization of 228 AA circulating in the form of small peptides. 229 In goats at early and late lactation, methionine, histidine, threonine, proline, and 230 phenylalanine in milk protein output were not accounted for in estimates of uptake of free 231 AA (Mabjeesh et al., 2002). An in vivo isotope kinetic technique was used to determine 232 the sources of AA for milk protein synthesis in which a long term (greater than 20 h) i.v. 233 infusion of a labeled AA (e.g., methionine) was performed until an isotopic steady state 234 was reached in the plasma-free AA pool and in the secreted milk casein. A dilution of 235 isotope was observed in milk casein indicating an alternate source of AA being used for 236 protein synthesis. The contribution of AA from intracellular protein turnover was 237 removed as a possibility due to the high fractional rate of protein synthesis in mammary 238 tissue and observation of complete turnover of proteins at the end of a 30-h infusion of 239 isotope. Mabjeesh et al. (2005) concluded that circulating unlabeled peptides contributed 240 to AA use for milk protein synthesis by mammary tissue with an estimated 7 to 18% of 241 methionine in casein being derived from peptides. When the estimate of methionine 242 derived from peptides was summed with an estimate of the contribution of free AA, total 243 uptake of methionine by the udder was in close balance with the estimate for milk output. 244 Tagari et al. (2004, 2008) demonstrated that peptide-bound AA represented a 245 important portion of total AA flux across the PDV and also represented a key 246 contribution to the mammary gland for milk protein synthesis. They reported, in the 2004 247 paper, that the PDV flux of total free essential AA was greater in cows fed steam-flaked 248 corn versus steam-rolled corn (571.2 vs. 366.4 g/12 h, respectively). The PDV flux of 249 essential peptide-bound AA was 69.3 ± 10.8 and 51.5 ± 13.2 g/12 h for cows fed steam- 11 Downloaded from jas.fass.org by on April 3, 2010. Journal of Animal Science Page 12 of 74 250 flaked or steam-rolled corn, respectively. Mammary uptake of essential peptide-bound 251 AA was greater for cows fed steam-flaked corn than steam-rolled corn (25.0 vs. 15.1 g/12 252 h, respectively). In the 2008 study in which processing effects of sorghum rather than 253 corn were evaluated, PDV flux of free AA was greater in cows fed dry-rolled as 254 compared with steam-flaked sorghum, while PDV flux of peptide AA was greater in 255 cows fed steam-flaked sorghum. In these cows milk production output did not differ 256 between the two dietary groups and the authors attributed this to a possible compensatory 257 effect of peptide-bound amino acids in the cows containing lower plasma levels of free 258 AA where peptide-bound AA could be used in response to the free AA pool shortage. 259 Tagari and colleagues suggested that peptide-bound AA may play a role in contributing 260 to AA for milk protein synthesis. With all of the accumulated data demonstrating the 261 presence of significant levels of circulating peptides in the blood, especially in ruminants, 262 and with the demonstration of significant stomach and intestinal absorption of AA in the 263 form of small peptides, peptide-bound AA in the blood may be important from a 264 nutritional perspective. 265 EVIDENCE FOR PEPTIDE TRANSPORT OUT OF ENTEROCYTES 266 Multiple research groups have independently identified a peptide transporter 267 expressed in the basolateral membrane of enterocytes that is responsible for movement of 268 peptides from inside the cell to the portal circulation. It has been suggested, though not 269 proven, that the enterocyte contains high concentrations of intracellular peptidases that 270 hydrolyze peptides into their constituent amino acids resulting in only free amino acid 271 transport out of the cell. Kim et al. (1972) detected peptidase activity in the brushborder 272 and soluble fractions of rat intestinal mucosa for 13 dipeptides and 5 tripeptides with the 12 Downloaded from jas.fass.org by on April 3, 2010. Page 13 of 74 Journal of Animal Science 273 soluble fractions constituting 80 to 90% and brushborder constituting 10 to 15%, of total 274 activity. The authors acknowledged that significant cross-contamination of each fraction 275 occurred and lability of the peptide hydrolases prevented further purification and 276 functional characterization of specific enzymes. While these data demonstrate the 277 presence of intracellular peptidases, they in no way demonstrate complete hydrolysis of 278 all dietary protein-derived peptides. Inside the cell peptides may either be hydrolyzed into 279 free AA by peptidases and transported across the basolateral membrane by free AA 280 transporters or intact peptides may be transported out of the cell by a peptide transporter. 281 The discovery of a peptide transporter in the basolateral membrane of enterocytes 282 (Terada et al., 1999) and the knowledge that some peptides are relatively resistant to 283 hydrolysis, provides at least presumptive evidence that a carrier-mediated mechanism 284 exists for transport of intact peptides to the bloodstream. In recent years, peptide transport 285 from inside the cell to the basolateral compartment has been studied to evaluate transport 286 characteristics of the basolateral peptide transporter. Irie et al. (2004) characterized the 287 efflux properties of the basolateral peptide transporter in Caco-2 cells by preloading cells 288 with [14C]-Gly-Sar and sampling the medium on the basolateral side after incubation. 289 Efflux to the basolateral side was not affected by basolateral pH and was saturatable with 290 a Kt of 24.8 ± 6.4 mM and Vmax of 1.61 ± 0.35 nmol/mg protein/min, indicating a low- 291 affinity. The Kt in the efflux direction was five to 10 times greater than in the influx 292 direction, indicative of asymmetry in substrate affinity. 293 The identity of the basolateral peptide transporter remains elusive. Shepherd et al. 294 (2002) identified a candidate protein in the basolateral membrane of rat jejunum through 295 the use of photoaffinity labeling with [4-azido-D-phe]-L-ala. They observed that 13 Downloaded from jas.fass.org by on April 3, 2010. Journal of Animal Science Page 14 of 74 296 perfusion of the label at the serosal side had no effect on transepithelial movement of a 297 non-hydrolyzable dipeptide D-phe-L-gln from the lumen, thus suggesting that [4-azido- 298 D-phe]-L-ala was unable to enter the mucosal side from the basolateral side. This is 299 supportive of a strongly asymmetric basolateral peptide transporter. However, when the 300 label was perfused luminally, there was a 40% reduction in the rate of transepithelial 301 transport of D-phe-L-gln and 60% increase in the accumulation of this dipeptide in the 302 mucosa indicating that the label enters the cell from the lumen and binds to the endofacial 303 side of the basolateral transporter and competes with and inhibits transepithelial 304 movement of D-phe-L-gln. The labeled candidate protein was extracted from SDS-PAGE 305 gels and analyzed by MALDI-TOF. Interestingly, database searches for the 112 kDa 306 candidate protein with a pI 6.5 revealed no similarities to PepT1 or other known proteins, 307 thus establishing that this is a novel protein. Identification and molecular characterization 308 of the basolateral peptide transporter will facilitate studies aimed at maximizing transport 309 of peptides and peptide-like drugs across the small intestine. 310 CLONING AND CHARACTERIZATION OF PEPT1 311 The peptide transporters are members of the Proton-coupled Oligopeptide 312 Transporter (POT) super-family, also called the Peptide Transporter (PTR) family 313 (Daniel and Kottra, 2004). Table 1 summarizes the currently identified members of the 314 peptide transporter family. In 1994, the first PepT1 mRNA was cloned from the rabbit 315 (Fei et al., 1994). The remainder of this review will focus on structural and functional 316 characteristics of PepT1 and the factors that regulate its expression including diet, 317 developmental stage, hormones, diurnal rhythm, and disease. 14 Downloaded from jas.fass.org by on April 3, 2010. Page 15 of 74 Journal of Animal Science 318 While results from many studies demonstrated uptake of peptides into intestinal 319 cells, the transporter responsible for peptide uptake was not identified until the 1990s. 320 The PepT1 (SLC15) was cloned by microinjecting messenger RNA isolated from rabbit 321 intestine into Xenopus oocytes resulting in functional expression of the protein, predicted 322 to consist of 12 transmembrane domains (TMD; Boll et al., 1994; Fei et al., 1994). The 323 first characterization of the peptide transporter in a livestock animal was reported by 324 Matthews et al. (1996) where sheep omasal RNA encoding for the peptide transporter 325 was expressed in Xenopus oocytes and dipeptide transport was demonstrated. They 326 observed that Gly-Sar uptake was saturatable (Kt = 0.4 mM), and inhibited 44% by 327 carnosine, 94% by methionylglycine, and 91% by glycylleucine, but not by free glycine. 328 The presence of mRNA that encodes for a peptide transporter in sheep omasal epithelia 329 was confirmed by Pan et al. (1997) when they demonstrated transport for di- and 330 tripeptides in oocytes injected with poly(A)+ RNA. 331 The peptide transporter, PepT1, has been cloned in multiple vertebrate species 332 including the rabbit, rat, mouse, sheep, chicken, turkey, dog, human, pig, cattle, monkey, 333 atlantic cod, and zebrafish (Table 2). The size of PepT1 ranges from 707 to 729 AA. 334 Expression and/or activity of PepT1 has been detected in other species including the 335 guinea pig (Himukai et al., 1983), hamster (Burston and Matthews, 1990), and the black 336 bear (Gilbert et al., 2007a). Peptide transporters have also been found in bacteria, yeast, 337 plants, and invertebrates (Daniel, 2004). Recently, a prokaryotic H+-dependent peptide 338 transporter, YdgR, was characterized and found to have features very similar to 339 mammalian PepT1 (Weitz et al., 2007). 340 SUBSTRATE SPECIFICITY OF PEPT1 15 Downloaded from jas.fass.org by on April 3, 2010. Journal of Animal Science Page 16 of 74 341 Potentially all 400 di- and 8,000 tripeptides can be transported by PepT1. It is 342 thought that water plays an important role in accommodating the broad specificity of 343 PepT1 by shielding the charges of the AA side chains within the substrate binding 344 domain of PepT1 which allows both charged and uncharged substrates to bind at the 345 same domain (Daniel, 2004). Metal ions (i.e., Zn2+, Mn2+, and Cu2+) also can interact 346 with transporter proteins to enhance peptide absorption (Leibach and Ganapathy, 1996). 347 PepT1 accepts pharmaceutical compounds with structural similarities to peptides 348 called “peptidomimetics” and participates in their absorption which is of major 349 therapeutic value (Leibach and Ganapathy, 1996; Brandsch et al., 2004). This allows for 350 the design of pharmacological drugs that possess acceptable oral availability because of 351 transport by PepT1. PepT1 and PepT2 have been found to transport cephalosporins, 352 penicillins, an aminopeptidase inhibitor bestatin, valine ester prodrugs of acyclovir and 353 ganciclovir, and inhibitors of angiotensin-converting enzyme, to name a few (Steffansen 354 et al., 2004). For an excellent review on the topic see Brodin et al. (2002). 355 Peptide transport was determined to be proton-dependent (Figure 1 A). Using the 356 two-microelectrode voltage-clamp technique in PepT1 cRNA-injected oocytes and 357 measuring currents for Gly-Sar transport, an inward H+ current was detected 358 demonstrating that peptide transport via PepT1 was proton-dependent among all species 359 tested (Adibi, 1997; Pan et al., 2001; Chen et al., 2002b; Klang et al., 2005; Van et al., 360 2005). The proton to substrate ratio for neutral and cationic AA transported by PepT1 is 361 one, whereas the ratio for charged anionic AA is two (Steel et al., 1997). The unstirred 362 water layer at the brushborder membrane is an isolated microenvironment free from the 363 influence of the luminal contents and maintains a high extracellular concentration of 16 Downloaded from jas.fass.org by on April 3, 2010. Page 17 of 74 Journal of Animal Science 364 protons (Adibi, 1997). Following uptake of peptides and H+ by PepT1, protons are then 365 transported out of the cell by the Na+/H+ exchanger on the brush-border membrane in 366 exchange for Na+. The Na+ in turn is taken out of the cell by the Na+/K+ ATPase pump on 367 the basolateral membrane where three Na+ are transported out of the cell in exchange for 368 two K+, restoring the electrochemical gradient. 369 Binding affinity for charged peptides and peptidomimetic drugs changes when pH 370 is altered (Daniel and Kottra, 2004). At brushborder membrane pH (5.5-6.0), neutral and 371 acidic peptides were preferred substrates for ovine PepT1 (Pan et al., 2001) and rabbit 372 and human PepT1 (Amasheh et al., 1997; Steel et al., 1997) when expressed in Xenopus 373 oocytes. Chen et al. (2002b) tested uptake of [3H]-Gly-Sar in CHO cells transfected with 374 chicken PepT1 and observed that uptake was greater at pH 6.0 and 6.5 than at 5.0, 5.5, 375 7.0, or 7.5 with similar results observed in oocytes expressing chicken PepT1. It is 376 important to point out though that pH dependence has been studied in vitro. In the intact 377 intestine, an acidic microclimate is maintained at the brushborder membrane independent 378 of the intestinal luminal contents (Daniel, 2004). Changes within the lumen do not 379 directly affect the unstirred water layer, and thus the results of in vitro studies are difficult 380 to relate to the intact, functioning intestine. 381 The affinity constants of peptide substrates for PepT1 vary widely from µM to 382 mM. Two methods have been employed to measure transport kinetics or binding affinity 383 of peptide substrates. The transport (Kt) of peptides is measured in Xenopus oocytes 384 expressing PepT1. The transport of the proton which is cotransported with the peptide is 385 measured by voltage clamping studies. Alternatively, the substrate affinity of PepT1 is 386 determined by an uptake inhibition assay. In this assay, the concentration of unlabeled 17 Downloaded from jas.fass.org by on April 3, 2010. Journal of Animal Science Page 18 of 74 387 peptide that inhibits 50% of labeled Gly-Sar uptake (IC50) is determined. In Table 3, the 388 Kt and IC50 values for sheep, chicken, and pig PepT1 are shown compared to human 389 PepT1. The Kt/IC50 values for Gly-Sar were similar for sheep (0.61 mM), chicken (0.47 390 mM), pig (0.94 mM), turkey (0.69 mM; Van et al., 2005), and human (1.2 mM) PepT1. 391 In contrast, the Kt values for the di and tripeptides tested ranged from 0.027 to 6.9 mM 392 and the IC50 values ranged from 0.005 to 7.9 mM. The reported IC50 values for human 393 PepT1 were higher than for sheep, pig, and chicken PepT1 and may reflect the different 394 cells used for the assays (MDCK cells for human PepT1 vs CHO cells for sheep, chicken 395 and pig PepT1). In general, tetrapeptides were poor substrates for PepT1 as indicated by 396 the IC50 values ranging from 0.95 to 27 mM or no response in the voltage clamp assays. 397 In most cases the IC50 values are comparable to the Kt values for sheep and chicken 398 PepT1. Peptides with two basic amino acids (Arg-Lys, Lys-Lys, Lys-Trp-Lys, Lys-Tyr- 399 Lys) were poorly transported, which suggests PepT1 can accommodate two positive 400 charges one from the peptide and one from the proton but not three positive charges. An 401 interesting species difference was seen with the dibasic Lys-Lys, which was a good 402 substrate for the ruminant sheep PepT1 but a poor substrate for the mongastric chicken, 403 pig, and human PepT1. The peptides that were used in these studies represented a variety 404 of molecular weights, net charges, and hydrophobicities, but there was no correlation 405 between any of these three parameters and affinity constants. It should also be pointed 406 out that although PepT1 is generally classified as a low-affinity transporter, affinity 407 constants for substrates can vary depending on the model system and transport conditions 408 used. In a review, Brodin et al. (2000) emphasized a point made by Meredith and Boyd 18 Downloaded from jas.fass.org by on April 3, 2010. Page 19 of 74 Journal of Animal Science 409 (2000) that affinity is the result of a “sum of a number of discrete interactions” and can 410 not be determined by a single parameter. 411 It should also be pointed out that there is competition among peptides for 412 transport (Matthews et al., 1979; Taylor et al., 1980; Pan et al., 2001; Klang et al., 2005) 413 and stimulatory effects of peptides on transport activity (Addison et al., 1974), thus 414 further complicating the relationship between peptide composition and transporter 415 affinity. Peptide composition may also influence the rate and extent of intracellular and 416 extracellular hydrolysis, as demonstrated by Adibi et al. (1986) when they observed that 417 rate of appearance of constituent AA comprising different dipeptides differed after 418 intravenous injection. 419 Vig et al. (2006) conducted a comprehensive study aimed at determining PepT1 420 substrate specificity and structural characteristics. They tested 73 peptides (mostly 421 dipeptides) for their binding affinity and ability to enhance transport activity of PepT1 in 422 MDCK cells using Gly-Sar as a reference substrate. Twenty one substrates, many of 423 which showed affinity for PepT1, did not activate PepT1 suggesting that they are not 424 substrates for transport. This finding contradicts the current mode of thought that all di- 425 and tripeptides serve as substrates for PepT1. Of these 21 non-transported substrates, Trp- 426 Trp was further evaluated for transport activity. Uptake of [3H]-Trp-Trp in MDCK-PepT1 427 transfected and MDCK-mock transfected cells showed similar kinetics, confirming that 428 Trp-Trp was not a substrate for transport. For peptides that did activate PepT1, Vig et al. 429 (2006) concluded that in general, N-terminal, large, hydrophobic AA enhanced PepT1 430 activity. In terms of charge, neutral AA were better substrates for PepT1. Aromaticity 431 enhanced activity and the authors suggested that the inability of Trp-Trp to be transported 19 Downloaded from jas.fass.org by on April 3, 2010. Journal of Animal Science Page 20 of 74 432 by PepT1 was the result of the large total volume of this dipeptide exceeding total 433 capacity of the binding pocket of the PepT1 protein. 434 There has been much effort to characterize the regions of the PepT1 protein 435 important in substrate recognition and affinity as well as proton binding. The N-terminal 436 half of PepT1 consisting of transmembrane domains (TMD) 1 to 6 is thought to contain 437 the AA residues associated with pH changes during peptide uptake and substrate binding 438 (Terada et al., 2000a), while the C-terminal AA (TMD 7 to 9) are thought to play an 439 important role in determining substrate affinity (Fei et al., 1998). Histidine residues are 440 important in the substrate binding domain of peptide transporters. Pretreatment of renal 441 brushborder membrane vesicles with DEPC, a histidine-modifying reagent, abolished 442 peptide transport (Meredith and Boyd, 2000). Mutations of human or rat PepT1 His-57 443 and rat His 121 (Fei et al., 1997; Terada et al., 1996) either markedly reduced or 444 abolished transport activity and were therefore considered to be extremely important for 445 PepT1 activity. Mutations of rabbit PepT1 His-57 also produced a non-functional 446 transporter and mutations of the tyrosine residues that flank rabbit His-57 (Tyr-56 or Tyr- 447 64) also produced a transporter with no activity (Chen et al., 2000). Immunostaining 448 revealed that transporter number on the plasma membrane was not impaired by His-57 or 449 His-121 mutation (Terada et al., 1996), indicating that protein function rather than 450 production was impaired. Mutations of rabbit His-121 resulted in reduced uptake and a 451 much lower affinity, suggesting a role in substrate recognition. The largest decline in 452 affinity occurred for anionic substrates, indicating that His-121 plays a role in protonating 453 negatively-charged substrates (Chen et al., 2000). It was then proposed that His-57 is 454 critical for H+-coupling and that the adjacent aromatic residues stabilize the positive 20 Downloaded from jas.fass.org by on April 3, 2010. Page 21 of 74 Journal of Animal Science 455 charge on the proton through cation- interactions. Doring et al. (2002) suggested that 456 rabbit PepT1 AA residues 1 to 59 are an important part of the substrate binding domain 457 and interact with side chains of substrates and AA residues 60 to 91 are important for the 458 pH profiles of peptide uptake, consistent with previous studies. Collectively, these studies 459 are in agreement with a molecular modeling approach to studying structure and function 460 of PepT1 where it was predicted that TMD 7 to 10 form part of a peptide transport 461 channel and TMD 1, 3 and 5 form the other half, with specific AA residues in each TMD 462 playing an important role in substrate binding (Bolger et al., 1998). 463 There are other various structural characteristics that have a marked effect on 464 peptide transport activity. Peptide transporters exhibit a greater affinity for peptides 465 containing the L-isomers of AA compared to the D-isomers (Leibach and Ganapathy, 466 1996; Brandsch et al., 2004). Terada et al. (2000b) demonstrated that dipeptides with a 467 modified -amino group, such as N-methyl-Gly-Gly and N-formyl-Met-Ala, showed 468 reduced affinity for PepT1 than their original counterparts (Gly-Gly and Met-Ala, 469 respectively). A peptide bond is not required for substrate recognition. Doring et al. 470 (1998) found that PepT1 and PepT2 recognized and transported substrates as simple as 471 omega-amino fatty acids with a positive amino and negatively-charged carboxyl head 472 group, which were separated by a minimum of four methylene groups. 473 TISSUE AND CELLULAR DISTRIBUTION OF PEPT1 474 With the cloning and characterization of PepT1, considerable efforts have been 475 made to evaluate its tissue and cellular distribution in the body and to relate the 476 localization of the transporter to the nutritional significance of small peptides. PepT1 477 mRNA and protein are expressed primarily in intestinal and renal epithelial cells (Daniel 21 Downloaded from jas.fass.org by on April 3, 2010. Journal of Animal Science Page 22 of 74 478 and Kottra, 2004). The renal peptide transporter, PepT2, in contrast to PepT1, is 479 expressed at greatest levels in the kidney tubule and to a lesser extent in the lung, 480 mammary gland, choroid plexus, and glia cells in the nervous system, but not in the 481 intestine (Daniel, 2004). Sites with lower levels of PepT1 expression and/or activity 482 include the colon, human bile duct epithelium, and rabbit brain and liver (Ford et al., 483 2003; Knutter et al., 2002; Miyamoto et al., 1996). Although almost no dietary peptides 484 or AA reach the colon, there is a large supply of endogenous proteins in the colon, 485 possibly serving as substrates for proteolysis by microflora. Intestinal-specific 486 expression of PepT1 is regulated by CDX2, a transcription factor that plays an important 487 role in the proliferation, differentiation, and maturation of intestinal epithelial cells 488 (Shimakura et al., 2006b). This regulation most likely occurs through interactions with 489 the ubiquitous Sp1 transcription factor for which there is a recognition site in the PepT1 490 promoter that controls basal activity (Shimakura et al., 2005). 491 Within the small intestine there are interesting species differences in PepT1 492 expression. Expression of PepT1 mRNA was detected in greatest quantities in the 493 duodenum, jejunum, and ileum of the chicken, pig, and ruminant, respectively (Chen et 494 al., 1999). These differences may correspond to differences in site of maximal digestion 495 and absorption. In the chicken, there was considerable expression of PepT1 in the ceca in 496 addition to the small intestine and kidney (Chen et al., 2002b). In rats, protein expression 497 was highest in the distal small intestine (ileum) as compared to the jejunum (Tanaka et 498 al., 1998). Howard et al. (2004) found that rat PepT1 mRNA was equally expressed 499 throughout the length of the small intestine. In the black bear, mRNA abundance of 500 PepT1 was greatest in the mid-region of the intestinal tract (Gilbert et al., 2007a). In 22 Downloaded from jas.fass.org by on April 3, 2010. Page 23 of 74 Journal of Animal Science 501 contrast to most species, ruminants, such as sheep and cattle, express PepT1 mRNA in 502 the stomach, specifically, in the omasum and rumen (Chen et al., 1999; Pan et al., 2001). 503 Expression of intestinal PepT1 increases with enterocyte maturation. In situ 504 hybridization has been used to examine cellular distribution of PepT1 mRNA. Freeman 505 et al. (1995) found that rabbit PepT1 mRNA staining was restricted to enterocytes 506 between the crypt-villus junction and villus tip with increased expression towards the tip. 507 Within the colon, expression was restricted to the surface columnar epithelial cells. 508 Immunohistochemical analyses showed that in rats, PepT1 protein was localized to the 509 brushborder membrane of cells lining the villi with increasing intensity from the crypt- 510 villus junction to the villus tip; no protein was detected in the crypts (Ogihara et al., 1996; 511 Tanaka et al., 1998). Interestingly, restriction of PepT1 to the brushborder membrane 512 may be developmentally regulated. Hussain et al. (2002) observed PepT1 protein at fetal 513 d 18 and day of birth in the duodenum; and right after birth, PepT1 spread to the 514 subapical cytoplasm, basal cytoplasm, and basolateral membrane in addition to the apical 515 membrane. At weaning and adulthood, PepT1 expression was restricted to the 516 brushborder membrane. Because its expression is mainly detected in the apical membrane 517 of small intestinal enterocytes lining the villi, PepT1, as a broad specificity, low-affinity, 518 high-capacity transporter, is strategically located to maximize assimilation of AA from 519 the diet. 520 DIETARY REGULATION OF PEPT1 521 Intestinal nutrient transport is regulated by diet and by substrate concentration in 522 the lumen. Ferraris and Diamond (1989) rationalized the differences observed in 523 transporter regulation among different nutrients. They suggested that transporters for 23 Downloaded from jas.fass.org by on April 3, 2010. Journal of Animal Science Page 24 of 74 524 metabolizable nontoxic nutrients, such as sugars, nonessential AA, and short-chain fatty 525 acids should be upregulated with increasing dietary level and that transporters for 526 essential nutrients, which are toxic in large quantities, such as water-soluble vitamins and 527 minerals should be downregulated by increasing concentrations of substrates. For AA, 528 the matter is complicated by the fact that AA can be also used as sources of energy. Some 529 AA are more essential to a cell than others, or more toxic. Furthermore, enterocytes 530 express transporters with overlapping substrate specificity and transporters that mediate 531 the movement of both essential and nonessential AA (Karasov et al., 1987). Thus, it 532 becomes difficult to predict whether a transporter should be upregulated in response to 533 certain AA deficiencies or imbalances. 534 Substrates can regulate transporters specifically and nonspecifically. Nonspecific 535 regulation involves general changes in mucosal surface area, transcellular 536 electrochemical gradient, paracellular permeability, and plasma membrane lipid 537 composition and fluidity (Ferraris, 1994, 2000). Specific regulation includes changes in 538 the site density of specific transporters in enterocytes as a result of changes in protein 539 synthesis or degradation rate or an increased insertion of preformed cytoplasmic 540 transporters into the brush border membrane (Ferraris and Diamond, 1989). 541 The influence of dietary protein on PepT1 expression and activity has been a 542 popular area of research in recent years. From a commercial livestock industry 543 perspective, dietary protein is a costly nutrient and even fractional improvements in its 544 utilization has the potential to save the industry millions of dollars as well as reduce 545 nitrogen excretion into the environment. Although dietary protein regulation of PepT1 546 expression and activity has not been as well studied in agriculturally important species, 24 Downloaded from jas.fass.org by on April 3, 2010. Page 25 of 74 Journal of Animal Science 547 much of the work conducted in biomedical species is broadly applicable and provides a 548 starting point for other studies aimed at determining the nutritional relevance of peptide 549 transport. 550 Our lab was the first to evaluate the effect of dietary protein quality on expression 551 of PepT1. Broiler chicks fed a diet containing a low quality protein (corn gluten meal) 552 showed decreased expression of PepT1 from d 3 to d 7 followed by an increase in 553 expression to d 14. In chicks that consumed an equal amount of the same diet substituted 554 with a higher quality protein (soybean meal), expression of PepT1 mRNA rose 555 continuously with age from d 3 to d 14 and birds were heavier (Gilbert et al., 2008). It 556 was thought that in birds consuming the soybean meal, continuous upregulation of the 557 transporter served as a mechanism to maximize assimilation of amino acids from a well- 558 balanced mixture, while in birds consuming the corn gluten meal, the effect was more 559 complicated due to the severely imbalanced diet. 560 In general, peptide transporter expression and activity increases with dietary 561 protein and peptide level. Shiraga et al. (1999) maintained rats on a 20% casein diet for 1 562 wk. A group of rats were switched to a protein-free diet and others were switched to diets 563 consisting of 50%, 20%, or 5% casein, 20% of a dipeptide, or 10% of a single AA, fed 564 for 3 d. Greater levels of dietary protein were associated with greater expression of 565 PepT1 mRNA and protein. Compared with rats fed the protein-free diet, there was an 566 increase in PepT1 mRNA and protein for rats fed the Gly-Phe diet and significantly 567 higher PepT1 mRNA for the phenylalanine-fed but not the glycine-fed rats. Results from 568 this study demonstrated that PepT1 may be regulated by specific amino acids and is very 569 responsive to changes in dietary protein, in particular, quantity and composition. 25 Downloaded from jas.fass.org by on April 3, 2010. Journal of Animal Science Page 26 of 74 570 A high-protein diet (72 vs. 18%) resulted in increased absorption of the dipeptide 571 carnosine in mice (Ferraris et al., 1988) and feeding a protein-free diet for 40 to 84 d 572 resulted in an increased ability of rat jejunum to transport a methionine dipeptide but not 573 free methionine (Lis et al., 1972). It is perhaps counterintuitive that both abundance and 574 lack of substrate will stimulate PepT1. Upregulation by a high-protein diet appears to be 575 a mechanism to take advantage of the abundant resource while upregulation in response 576 to a lack of substrate may be a compensatory mechanism to scavenge amino acids in the 577 lumen. The magnitude of the response in changes of PepT1 expression and activity will 578 probably be dependent on length of time for a particular dietary manipulation, availability 579 of transportable substrate, amino acid composition (concentrations of free and peptide- 580 bound), and presence of other components in the lumen that can change digestive and 581 absorptive dynamics (i.e., sugars, vitamins, minerals, fats, etc.). 582 In chickens, an increase in PepT1 mRNA was observed in the intestine of 583 chickens fed 18 and 24% crude protein (CP) diets with restricted food intake and a 584 decrease in PepT1 mRNA was observed in chickens fed a 12% CP diet (Chen et al., 585 2005). In chicks fed the 24% CP diet ad-lib, there was lower expression of PepT1 as 586 compared with chicks consuming restricted amounts of the diet. Thus, the increase in 587 PepT1 was probably not due to an increase in CP but instead to the feed restriction. 588 Similarly, Gilbert et al. (2008) found that feed restriction increased expression of PepT1 589 mRNA from d 3 to d 14 posthatch in broilers. 590 Regulation of PepT1 by dietary substrate appears to occur by two mechanisms: 1) 591 by increasing mRNA stability and 2) by increasing gene transcription rate (Adibi, 2003). 592 Upregulation of PepT1 in response to low-protein or protein-free diets may be a 26 Downloaded from jas.fass.org by on April 3, 2010. Page 27 of 74 Journal of Animal Science 593 mechanism to compensate for reduced surface area as protein malnutrition is known to 594 cause significant reductions in intestinal surface area and mucosal mass (Ferraris and 595 Carey, 2000). In protein-malnourished rats (5% CP diet), intestinal villi were shorter in 596 comparison with control rats (20 % CP diet). There were older enterocytes on villi in 597 malnourished rats compared with those at the same villus height in well-fed rats. Hence, 598 the mechanism leading to a fasting or malnutrition-related increase in nutrient transport 599 may be a combination of increased gene expression and ratio of transporting to non- 600 transporting cells (Ferraris and Carey, 2000). 601 The mechanism of dietary protein regulation of nutrient transporters may be 602 through a pathway of direct substrate regulation. Shiraga et al. (1999) found elements in 603 the 5` upstream region of rat PepT1 responsive to peptides and free AA. Fei et al. (2000) 604 identified a similar amino acid responsive element in the promoter region of mouse 605 PepT1. To study responses of the PepT1 gene to specific amino acids or peptide 606 substrates, in-vitro experiments using cultured cells that express PepT1 were conducted. 607 This method establishes a controlled model for observing the impact of a single substrate 608 on regulation of the PepT1 gene. Addition of Gly-Sar to the medium of Caco-2 cells 609 caused a threefold and twofold increase in the expression of human PepT1 mRNA and 610 protein, respectively (Thamotharan et al., 1998). Treatment with brefeldin, an inhibitor of 611 protein transport from the endoplasmic reticulum to the Golgi, abolished transport, 612 showing that increased synthesis and processing by the trans Golgi network accounted for 613 increased expression at the apical membrane. Similar uptake experiments were performed 614 with a naturally occurring dipeptide, Gly-Gln, confirming the physiological relevance of 615 these findings (Walker et al., 1998). These results demonstrate direct regulation of PepT1 27 Downloaded from jas.fass.org by on April 3, 2010. Journal of Animal Science Page 28 of 74 616 by its own substrate which has important implications for nutritional supplements aimed 617 at improving AA uptake. Additionally, it is worth noting that free AA uptake may be 618 indirectly regulated by PepT1 activity. Since many AA transporters serve as exchangers, 619 filling of cells with a variety of AA as peptides by PepT1 may be important for net 620 movement of AA. For example, Wenzel et al. (2001) demonstrated that uptake of 621 dipeptides caused stimulation of AA uptake by the bo,+ system. 622 Short-term feed deprivation is common in domestic animals. In cattle and pigs it 623 can occur under conditions such as weaning and relocation and in chickens it is common 624 for a delayed access to feed for 48 h or more after hatch to occur due to bird processing 625 and transport (Noy and Sklan, 1998, Vieira and Moran, 1999; Bigot et al., 2003). Short- 626 term starvation in rats has been shown to increase mRNA and protein expression of 627 PepT1 (Ihara et al., 2000). Rats starved for 4 d exhibited a 179% increase in mRNA and 628 protein expression. Rats that were fed 50% of the intake of controls for 10 d and rats 629 given total parenteral nutrition (TPN) for 10 d exhibited a 161% and 164% increase in 630 PepT1 mRNA, respectively. This dramatic upregulation occurred in spite of the fact that 631 the mucosal weight decreased in the starved and TPN group by 41 and 50%, respectively. 632 In a different study, PepT1 mRNA and protein increased threefold and the rate of peptide 633 transport in rats increased dramatically after only 1 d of fasting (Thamotharan et al., 634 1999a). Ferraris and Diamond (1989) described the regulation of nutrient transporters as 635 a way to match uptake capacity to requirements without wasting energy on unnecessary 636 transporters. 637 Howard et al. (2004) examined the effects of TPN and administration of 638 glucagon-like peptide (GLP-2) on the mRNA expression of PepT1 in rat small intestine. 28 Downloaded from jas.fass.org by on April 3, 2010. Page 29 of 74 Journal of Animal Science 639 Total parenteral nutrition for 7 d upregulated PepT1 mRNA in the distal intestine while 640 proximal (duodenal) mRNA was unchanged. Administration of GLP-2 inhibited the 641 effect of TPN on mRNA expression of PepT1. It is known that GLP-2 is a trophic factor 642 that maintains cellular protein synthesis during luminal starvation and perhaps infusion of 643 GLP-2 reduced the need for upregulation of PepT1. During luminal starvation, the need 644 to absorb endogenous protein products increases and thus, apical membrane transporters, 645 which in this study included NBAT, EAAC1, ASCT2, and PepT1, were upregulated in 646 the distal intestine to maximize assimilation of AA (Howard et al., 2004). 647 Peptide transporter expression and activity may also be altered by diurnal rhythm 648 (Pan et al., 2002, 2003, 2004). To date, studies involving this phenomenon have been 649 conducted in rodents which are nocturnal mammals. In rats, PepT1 expression increased 650 at night, paralleling the normal feeding behavior of these rodents. Interestingly, fasting or 651 an imposed daytime feeding abolished this pattern and expression patterns quickly 652 changed to accommodate the nutritional needs of the gut (Pan et al., 2004). This hints to 653 the plasticity of peptide transporter expression and how quickly the intestine is able to 654 adapt in response to environmental changes. This has important implications across 655 mammalian and avian species that can vary dramatically in feeding behavior. 656 Increased expression and activity of PepT1 during feed deprivation or restriction 657 may serve as a mechanism to compensate for reduced mucosal surface area. Delayed 658 access to feed for 36 h posthatch resulted in depressed villus height and crypt depth and 659 reduced growth in all intestinal segments (Uni et al., 1998). Silva et al. (2007) subjected 660 male broiler chicks to a feed restriction at 30% of ad libitum intake from 7 to 14 d and 661 found that feed restriction decreased the surface area of the tip of the enterocytes in the 29 Downloaded from jas.fass.org by on April 3, 2010. Journal of Animal Science Page 30 of 74 662 small intestine at 14 d. Ihara et al. (2000) found that starvation or TPN for 4 d in rats 663 were accompanied by reductions in mucosal weight by 41 and 50 %, respectively. Feed 664 restriction causes reduced absorptive surface area due to intestinal mucosal atrophy, 665 which may explain why peptide transporter gene expression and protein activity is 666 increased. As we begin to understand the factors that control expression of PepT1 and 667 discover how changes in the diet can manipulate these factors to our advantage, we can 668 begin to effect changes in the phenotype (BWG, feed conversion, milk yield, etc.) 669 through dietary manipulation of gene expression. 670 Shimakura et al. (2006a) proposed a mechanism for induction of intestinal PepT1 671 during situations of fasting or starvation. They demonstrated that PepT1 induction is 672 mediated by peroxisome-proliferator-activated receptor alpha (PPAR ), a member of a 673 family of nuclear receptors activated by fatty acid ligands, thus playing an important role 674 in the adaptive response to starvation. In PPAR -null mice, the fasting-induced 675 expression of PepT1 was abolished; whereas, in wild-type mice there was significant 676 increases in PPAR and PepT1 after 48 h of fasting. When the PPAR ligand WY- 677 14643 was orally gavaged to fed rats for 5 d, expression of PepT1 mRNA increased and 678 when Caco-2 cells were treated with the same ligand, expression of PepT1 increased and 679 uptake of [3H]-Gly-Sar increased. There was no increase in PepT1 expression or peptide 680 uptake when ligands specific for PPAR or PPAR / were administered to cells. 681 Although the functional response element or other regulatory region was not determined, 682 the authors suggested that PepT1 is either directly regulated by PPAR through binding 683 to a regulatory region or that PPAR induces expression of transcription factors, such as 684 SP1 or CDX2, which were also upregulated in this study in response to fasting. 30 Downloaded from jas.fass.org by on April 3, 2010. Page 31 of 74 Journal of Animal Science 685 DEVELOPMENTAL AND HORMONAL REGULATION OF PEPT1 686 Changes in growth, metabolism, and levels of circulating hormones may 687 influence intestinal peptide transport. Among hormones that have been demonstrated to 688 affect expression and/or activity of the peptide transporter are insulin (Thamotharan et al., 689 1999b), leptin (Buyse et al., 2001), epidermal growth factor (Nielsen et al., 2001, 2003), 690 and thyroid hormone, T3 (Ashida et al., 2002). To describe each of these hormones in 691 detail would be beyond the scope of this review; hence, we will only discuss insulin and 692 leptin as these may be most relevant from a nutritional perspective. Insulin is not a 693 hormone located in the gut lumen, but during normal physiological conditions, 694 circulating insulin can bind to receptors located on the basolateral membrane of 695 enterocytes (Adibi, 2003). Addition of 5 nM insulin to Caco-2 cells increased the number 696 of PepT1 transporters and stimulated Gly-Gln uptake (Thamotharan et al., 1999b). 697 Treatment of the basolateral membrane with 50 ng/mL insulin for 1 h increased Gly-Sar 698 uptake, whereas treatment of the apical membrane had no effect (Nielsen et al., 2003). 699 Leptin, a hormone that suppresses appetite and increases metabolism, is secreted by both 700 adipocytes and by the stomach and has been found to be released into the stomach and 701 reach the intestine in a nondegraded form where there are apical leptin receptors on 702 enterocytes (Buyse et al., 2001). Addition of leptin to only the apical membrane of Caco- 703 2 cells or mouse jejunum increased cephalexin and Gly-Sar transport and increased 704 membrane PepT1 while reducing intracellular quantities. Addition to only the basolateral 705 membrane had no effect on transport or PepT1 expression. Hindlet et al. (2007) 706 demonstrated that in leptin deficient ob/ob mice there was impaired peptide transport 707 activity (50% less Gly-Sar uptake as compared with wild-type mice) and gene expression 31 Downloaded from jas.fass.org by on April 3, 2010. Journal of Animal Science Page 32 of 74 708 (40% and twofold less PepT1 protein and mRNA, respectively, as compared with wild- 709 type mice) that was restored upon 7 d of continuous subcutaneous administration of 710 leptin. Stimulation of peptide transport by insulin or leptin is thought to involve increased 711 trafficking of cytoplasmic PepT1 proteins to the apical membrane. 712 Peptide transport activity varies with age (Leibach and Ganapathy, 1996), and 713 dietary changes during the prenatal, suckling, or weaning period may have irreversible 714 effects on nutrient transport that carryover into adulthood (Karasov et al., 1985; Pacha, 715 2000). Early ontogenetic development of the gut is characterized by morphogenesis and 716 cytodifferentiation during fetal development at which time the intestine becomes 717 prepared for postnatal life when it will assume complete responsibility for nutrient 718 absorption (Puchal and Buddington, 1992; Pacha, 2000). Prenatal expression of glucose, 719 peptide, and AA transporters was reported to be present in humans, guinea pigs, sheep, 720 rabbits, and rats (Guandalini and Rubino, 1982; Pacha, 2000; Shen et al., 2001). 721 At birth, the intestine becomes the site of nutrient assimilation and the animal 722 begins to consume a high-protein milk diet. At weaning, the animal shifts to the adult diet 723 consisting of predominantly carbohydrates. Xiao (2006) conducted one of few studies 724 involving developmental regulation of peptide and AA transporters in a domestic animal. 725 This study investigated the development of nutrient transporter expression during the two 726 most rapid stages of intestinal development: 1) at birth when the pig shifts diet from the 727 amniotic fluid to mother’s milk and 2) at weaning when the pig shifts diet from the 728 mother’s milk to a solid diet (Pacha, 2000). During suckling (d 0 to d 21) and 729 postweaning (d 21 to d 35), pig intestinal PepT1 mRNA remained constant except for a 730 decline one day after birth and a peak one day after weaning. Protein expression of PepT1 32 Downloaded from jas.fass.org by on April 3, 2010. Page 33 of 74 Journal of Animal Science 731 generally decreased with age in the duodenum and increased with age in the jejunum and 732 ileum (Xiao, 2006). Protein and mRNA expression of PepT1 decreased in all segments 733 during the first few days of suckling in contrast to several brushborder AA transporters 734 and peptide hydrolases that increased, suggesting a more complete hydrolysis of peptides 735 in the lumen and less availability of peptides during this time period. 736 Other mammals show similar changes in expression of PepT1 that correlate with 737 changes in the diet and intestinal development. Rat intestinal PepT1 mRNA and protein 738 was present at fetal d 20, increased at birth, and reached maximal expression levels 739 during d 3 to d 5 (Shen et al., 2001). The PepT1 mRNA levels then dropped to about 12% 740 of maximal levels by d 14 and rose to 23 to 58% of maximal expression levels at 741 weaning, after which expression plateaued. Similarly, PepT1 protein dropped sharply 742 after d 5, rose to 59 to 88% of maximal expression at weaning, and then plateaued at 743 adulthood (d 75). Colonic PepT1 mRNA and protein was detected at d 1 to d 5 and 744 dropped to almost undetectable levels at d 7 and was undetectable afterwards at all days. 745 The ability of the colon to transport peptides was suggested to serve as a compensatory 746 mechanism for the temporary low capacity for nutrient absorption in the small intestine. 747 The developmental regulation of ovine PepT1 in the dorsal rumen, ventral rumen, 748 omasum, duodenum, jejunum, and ileum of lambs was studied at 2, 4, 6, and 8 wk of age 749 (Poole et al., 2003). Ovine PepT1 was present in all tissues examined at 2 wk of age and 750 was not influenced by age. Since expression was evaluated starting at 2 wk, there may 751 have been earlier changes occurring; for example, induction by suckling as reported in 752 other species. Similarly, the last sampling date was 8 wk and lambs were not weaned in 753 this study, a dietary shift that induces changes in the transporter in other mammals. Poole 33 Downloaded from jas.fass.org by on April 3, 2010. Journal of Animal Science Page 34 of 74 754 et al. (2003) observed that lambs allowed to nurse but not allowed access to a creep-diet 755 at birth expressed greater quantities of PepT1 mRNA in the rumen. Because of the lack 756 of luminal substrate (i.e., the presence of the reticular groove should prevent milk from 757 entering the rumen) to act as inducers of expression, it was suggested that blood-derived 758 factors may be involved in the developmental regulation of ovine PepT1. 759 The turkey and chicken, although exhibiting a different mode of embryological 760 development than mammalian species, exhibit a similar developmental regulation of 761 PepT1 to prepare the intestine for immediate uptake of nutrients at hatch. In the bird 762 however, the diet shifts from the lipid-rich yolk as the main source of nutrients during 763 embryological development to a carbohydrate- and protein-rich diet posthatch. Van et al. 764 (2005) observed a 3.3-fold increase in mRNA expression levels of PepT1 in turkey 765 intestinal tissue from 5 d before hatch to day of hatch which is similar to rats in which 766 PepT1 mRNA spiked at birth (Shen et al., 2001). Chen et al. (2005) observed a 14- to 50- 767 fold increase in intestinal chicken PepT1 mRNA from embryo d 18 to day of hatch, with 768 expression peaking right before hatch. More recently, Gilbert et al. (2007b) conducted a 769 comprehensive study of the developmental regulation of peptide, AA, and sugar 770 transporter mRNA in the small intestine of two genetically-selected lines of broiler chicks 771 from embryo d 18 to d 14 posthatch. For all brushborder membrane transporters 772 evaluated, mRNA abundance increased with age with dramatic induction from embryo d 773 18 to day of hatch. It will be of interest to determine if embryonic nutritional modulations 774 (e.g., in-ovo nutrient administration) have effects on the developmental timing of PepT1 775 gene expression. Expression of PepT1 mRNA increased with age with greatest 776 expression at d 14 posthatch suggesting that the capacity for peptide absorption in 34 Downloaded from jas.fass.org by on April 3, 2010. Page 35 of 74 Journal of Animal Science 777 broilers continues to increase with maturity. Interestingly, in that same study, it was 778 observed that out of 14 transporter genes evaluated for mRNA expression, only PepT1 779 was influenced by genetic line. There was an approximately twofold difference in PepT1 780 mRNA suggesting a greater capacity for absorption of peptide-bound AA. While 781 transporter expression during development may be controlled by genetic determinants, 782 expression is also controlled at the transcriptional levels by many factors, including the 783 diet, suggesting that developmental programming is not absolutely predetermined (Pacha, 784 2000). 785 REGULATION OF PEPT1 DURING DISEASE 786 PepT1 exhibits an amazing resiliency in the intestine by maintaining expression 787 and activity levels in spite of intestinal damage. In the livestock industry this can have 788 important implications due to the prevalence of a variety of infections that plague the gut 789 and impair animal performance, cause mortalities, and result in substantial losses to the 790 producer. In this review we will focus only on the effects of colonic inflammation, 791 bacterial endotoxins, and parasitic infections on PepT1 gene expression, as these 792 situations are relevant to situations encountered in the livestock and poultry industry. The 793 effect of various pathogenic agents or parasites on peptide transporter expression is 794 dependent on the species, age, health status, site of infection in the gut, and duration of 795 exposure and infection, to name a few. 796 PepT1, which is expressed in very small quantities in the colon compared to the 797 proximal intestine (Ford et al., 2003), is upregulated in the colon of patients suffering 798 from short-bowel syndrome (SBS; Ziegler et al., 2002), perhaps serving as a mechanism 799 to maximize absorption of dietary AA in patients with a restricted capacity (Ziegler et al., 35 Downloaded from jas.fass.org by on April 3, 2010. Journal of Animal Science Page 36 of 74 800 2002). This same pattern of expression is also exhibited in patients suffering from chronic 801 ulcerative colitis and Crohn’s disease (Merlin et al., 2001). Also, N-formyl-methionyl- 802 leucyl-phenylalanine (fMLP) a major proinflammatory peptide of the human colonic 803 lumen is found at higher levels in the colon where bacterial loads are higher compared to 804 the small intestine and where PepT1 is normally minimally expressed. However, during a 805 diseased state PepT1 expression increases in the colon, transports fMLP, and stimulates 806 expression of MHC-1 genes (Merlin et al., 2001). Interestingly, leptin is increased in 807 inflamed colonic mucosa similar to PepT1. Nduati et al. (2007) demonstrated that leptin 808 induces colonic expression of hPepT1 via the transcription factors CREB and CDX2. 809 Bacterial infections caused by endotoxin administration in rats were shown to 810 regulate expression and activity of PepT1 in the intestinal tract (Shu et al., 2002). 811 However, in contrast to the other types of diseases discussed, endotoxin treatment served 812 to downregulate PepT1 mRNA and protein (32 to 62 % of controls) in rats, most likely 813 through the action of proinflammatory cytokines (IL-1 and TNF- ) that are increased by 814 lipopolysaccharide treatment. Sekikawa et al. (2003) found that infection with the 815 nematode Nippostrongylus brasiliensis in rats downregulated mRNA and/or protein 816 expression levels of GLUT5, PepT1, LAT2, and SGLT1 7 to 14 d after infection. This 817 downregulation of nutrient transporters could contribute to the malnutrition that ensues in 818 patients with a nematode infection (Sekikawa et al., 2003). 819 Barbot et al. (2003) examined the effects of Cryptosporidium parvum, a cause of 820 diarrhea, on PepT1 in rat intestine from d 4 to d 50. Rats were gastrically infused with C. 821 parvum on d 4 and the parasite disappeared on d 21. On d 10, the parasitic infection was 822 greater in the ileum than in the proximal small intestine. Villus atrophy occurred 36 Downloaded from jas.fass.org by on April 3, 2010. Page 37 of 74 Journal of Animal Science 823 throughout the small intestine, but was most pronounced in the ileum with villus 824 morphology returning to normal on d 21. PepT1 mRNA levels increased on d 10, during 825 the peak of the infection, and returned to normal levels after removal of the parasite (d 826 21). In the control animals, PepT1 mRNA was evenly distributed throughout the small 827 intestine from d 4 to d 50. Immunohistochemical staining revealed expression of PepT1 828 protein from d 4 to d 50 on the brushborder membrane throughout the small intestine, but 829 during the peak of infection (d 10 through d 12), PepT1 was also detected intracellularly, 830 after which it disappeared. This intracellular staining was not detected in the control 831 animals. It is not clear how the parasite may regulate PepT1 expression. One theory is 832 that it may interfere with trafficking of the protein from the cytoplasm to the membrane 833 (Barbot et al., 2003). 834 ALTERNATIVE MECHANISMS OF PEPTIDE UPTAKE 835 In thinking about peptide absorption in the gut through PepT1, it is important to 836 consider alternate routes of nutrient delivery to the cell, including paracellular (e.g., 837 tight-junctions) and non-transporter-mediated transcellular (e.g., endocytosis or diffusion) 838 uptake, especially if these processes not requiring input of energy by the cell can be 839 modulated through the diet (Figure 1 B and C). In earlier studies of peptide transport in 840 the ruminant, Matthews and Webb (1995) and McCollum and Webb (1998) alluded to 841 paracellular transport as a potential route of peptide uptake in the ruminant. Madara and 842 Pappenheimer and Reiss (1987) showed that mediated uptake of a substrate (e.g., 843 peptides via PepT1) was a prerequisite for paracellular uptake. In normal rats that 844 ingested a 5% glucose solution containing [123I]-labeled octapeptides of D-amino acids, 845 assumed to be resistant to hydrolysis and to not be a substrate for the peptide transporter, 37 Downloaded from jas.fass.org by on April 3, 2010. Journal of Animal Science Page 38 of 74 846 there was as much as 50% of the peptide excreted into the urine intact (Pappenheimer et 847 al., 1994). This suggests that transcellular Na+-dependent glucose transport triggers 848 dilation of the tight junctions permitting absorption of other solutes through solvent drag 849 (Pappenheimer and Reiss, 1987). Chediack et al. (2006) reported paracellular uptake of 850 two D-dipeptides, serine-aspartic acid and serine-lysine, in the house sparrow gut and 851 negligible activity of the peptide transporter. 852 In species where the permeability of tight junctions prevents significant passive 853 epithelial transport, there may be strategies for overcoming this barrier. Motlekar et al. 854 (2006) reported efforts to enhance uptake of the low molecular weight heparin, ardeparin, 855 through the use of zona occludens toxin (Zot), which is an enterotoxin obtained from 856 Vibrio cholerae. This toxin was shown to reversibly and safely open tight junctions. They 857 demonstrated that in the presence of 100 µg/kg of AT1002, a novel synthetic hexapeptide 858 derived from Zot, oral bioavailability of ardeparin was improved in the rat with no 859 detectable cytotoxicity or morphological damage to intestinal cells. Paracellular uptake 860 of peptides or peptidomimetics may also be influenced by bioactive components in the 861 diet. Capsaicin, the chemical responsible for the pungent properties of hot peppers, was 862 shown to reduce uptake of cephalexin in the jejunum and ileum of rats, primarily due to a 863 decrease in transcellular transport mediated by PepT1 (Komori et al., 2007). 864 Interestingly, capsaicin increased the paracellular permeabililty of cephalexin. These 865 results indicated that the transcellular and paracellular transport of peptides can be 866 independently altered. 867 An additional alternate route of peptide uptake could be through the non-carrier 868 mediated transcellular route. In recent years there has been much research focused on 38 Downloaded from jas.fass.org by on April 3, 2010. Page 39 of 74 Journal of Animal Science 869 cell-penetrating peptides (CPP) as routes of delivery for pharmacological substances. 870 Cell-penetrating peptides are peptides that are capable of translocating across the plasma 871 membrane and may at the same time deliver cargo to the interior of the cell (Palm et al., 872 2007) including oligonucleotides (Morris et al., 1997), peptide nucleic acids (Eriksson et 873 al., 2002), plasmids (Morris et al., 1999), proteins (Saalik et al., 2004), and liposomes 874 (Torchilin et al., 2001). Hallbrink et al. (2001) demonstrated that four different CPP were 875 capable of translocating a labeled pentapeptide into Bowes human melanoma cells. The 876 mechanism of how CPP are able to translocate the plasma membrane is still unclear. This 877 may occur through direct penetration of the plasma membrane or by various types of 878 endocytosis followed by endosomal release. Translocation may be initiated by binding of 879 cationic moieties on the CPP to negative charges on the plasma membrane. Sai et al. 880 (1998) demonstrated that intestinal absorption of a novel fluorescence-derivatized 881 cationic peptide 001-C8 (H-MeTyr-Arg-MeArg-D-Leu-NH(CH2)8NH2) occurred by 882 adsorptive-mediated endocytosis which occurs through binding of the cationic moieties 883 of the peptide to negative charges on the plasma membrane. The cationic peptide Tat, 884 derived from the protein transduction domain of HIV-1, was reported to enter cells by 885 macropinocytosis (Kaplan et al., 2005) or by binding to heparin sulfate receptors and 886 entering by either caveolar endocytosis (Fittipaldi and Giacca, 2005) or clathrin-mediated 887 endocytosis (Richard et al., 2005). Cell penetrating peptides could serve as a novel 888 nutrient delivery agent to overcome the impenetrability of the cell barrier. It would also 889 be of interest to further explore the possibility of delivering larger peptides that are not 890 transported by PepT1 to the interior of the cell. 891 DIETARY PEPTIDES 39 Downloaded from jas.fass.org by on April 3, 2010. Journal of Animal Science Page 40 of 74 892 Small peptides have been considered as a source of AA in feeding solutions or 893 diets for patients with altered intestinal absorptive function and food allergies (Clemente, 894 2000). Peptides offer an advantage over free AA that are unstable or insoluble and may 895 represent a more cost-effective way of supplementing AA (Zimmerman et al., 1997; 896 Lindemann et al., 2000; Dabrowski et al., 2003). Because of the labile nature of 897 glutamine and insoluble nature of tyrosine or tryptophan, absorption in a small peptide 898 form increases the availability of these AA to the body (Adibi, 1997). Also, the use of 899 dipeptides reduces the hypertonicity that results from a free AA feeding solution. 900 In all animal species evaluated thus far, there appears to be considerable capacity 901 in the small intestine for the absorption of amino acids in the form of small peptides. 902 Thus, it is reasonable to predict that incorporation of small peptides or hydrolyzed 903 proteins into the diet would exploit this ability and potentially enhance animal growth 904 and development. Additionally, di- and tripeptides are absorbed quickly and efficiently 905 by the intestine without initial pancreatic digestion (Zambonino Infante et al., 1997). 906 There are some reports of feeding hydrolyzed proteins to fish, which express high 907 levels of PepT1 in the small intestine (Verri et al., 2003; Ronnestad et al., 2007). Weight 908 gain and survival rate were improved for sea bass larva fed 20 or 40% of total nitrogen as 909 peptides, respectively, as compared with those fed only fish meal (Zambonino Infante et 910 al., 1997). Similarly, survival, final weight, total biomass, and malformation rates of sea 911 bass or carp larvae were optimal when 52% CP diets were fed that contained equal 912 amounts of yeast and fish protein hydrolysate as contributors of CP as compared with 913 equal parts of yeast and soy protein or fish meal (Cahu et al., 1998). In sea bass larvae fed 914 fish meal or fish meal plus hydrolysate as 25% of total nitrogen, weight gains were 40 Downloaded from jas.fass.org by on April 3, 2010. Page 41 of 74 Journal of Animal Science 915 similar in both groups (Cahu et al., 1999). It can be inferred from these studies that 916 substitution of fish protein hydrolysate for fish meal can improve performance of sea bass 917 or carp larvae. In these studies the fish meal hydrolysate was not produced from the same 918 fish meal to which it was compared, thus, these results must be considered preliminary. 919 In a more recent study, sea bass larvae were fed diets containing either 10% of a 920 sardine by-product hydrolysate (SH; 42% CP; main fraction of peptides between 200 to 921 500 Da), a commercial enzymatic hydrolysate (CPSP; 82% CP; main fraction of peptides 922 between 500 to 2500 Da), or 19% of either of the two hydrolysates (Kotzamanis et al., 923 2007). The remainder of the protein in the diet was supplied by fishmeal. The SH 924 hydrolysates were less soluble and contained a larger proportion of di- and tripeptides 925 than CPSP. Kotzamanis et al. (2007) observed that the 10% incorporation of CPSP 926 yielded the best growth and survival rates and intestinal development of the larvae as 927 indicated by early induction of brushborder membrane enzyme activites. This group also 928 appeared to have improved immunological status as indicated by lower levels of Vibrio 929 spp. in the larvae. The poorest performance data were obtained with the diet containing 930 10% of SH. Both the molecular weight distribution and concentration of dietary peptides 931 may influence growth performance and immunological development. The use of different 932 protein sources, hydrolysis conditions, and dietary concentrations makes it very difficult 933 to compare across studies and make claims regarding efficacy. The differences in 934 substrate affinity of PepT1 for the different peptides, the effect of peptides on gene 935 expression, as well as the 8,400 possible di- and tripeptides creates a challenge for the 936 nutritionist in providing a peptide profile that accommodates the digestive enzyme and 937 transporter physiology in the gut. 41 Downloaded from jas.fass.org by on April 3, 2010. Journal of Animal Science Page 42 of 74 938 Although a variety of peptide-based products are currently on the market, there 939 has been some debate over their efficacy due to the reasons already described. 940 Technological advances in amino acid analysis of peptide profiles will improve the 941 design of future feeding studies involving dietary protein replacement or supplementation 942 with peptides. Promising results have been obtained in studies involving pigs. Dried 943 porcine solubles (DPS) is a peptide product consisting of partially hydrolyzed residues 944 from porcine intestine (Nutra-Flo® Company, Sioux City, IA). Growth performance was 945 improved in weaned pigs that consumed diets containing DPS (Zimmerman et al., 1997; 946 Lindemann et al., 2000; DeRouchey et al., 2003). Additionally, an enzymatically digested 947 protein product produced from a proprietary blend of swine blood, selected poultry 948 tissues, and hydrolyzed feathers (Griffin Industries, Inc., Cold Spring, KY), was observed 949 to be similar in feeding value to menhaden fish meal, spray-dried animal blood cells, and 950 spray-dried plasma protein in early-weaned pigs. 951 CONCLUSIONS AND IMPLICATIONS FOR FUTURE RESEARCH 952 The intestinal peptide transporter, PepT1, is predominantly expressed in the small 953 intestine across species and mediates the transport of di- and tripeptides as well as 954 peptidomimetic compounds. Peptide transporter gene regulation has been researched 955 extensively and currently it is accepted that PepT1 is regulated by diet, developmental 956 stage, hormones, and disease. The mechanisms underlying changes in gene expression 957 are still unclear, but most likely involve changes in transcription rate, mRNA stability, 958 protein synthesis rate, and/or protein trafficking. 959 While practical applications of peptides to livestock nutrition are lacking, we hope 960 that the readers will exploit the information presented herein in order to stimulate further 42 Downloaded from jas.fass.org by on April 3, 2010. Page 43 of 74 Journal of Animal Science 961 investigations the results of which may enable improved nutritional management 962 decisions. Providing a diet that best accommodates the profile of digestive enzymes and 963 nutrient transporters present in the gut will result in improved utilization of dietary 964 protein, reduced nitrogen excretion into the environment, improved health, and improved 965 growth. The future of dietary peptides in livestock diets remains unclear, but we hope that 966 this review has served the purpose of enlightening some readers to embrace the concept 967 of peptide absorption and better understand the factors that contribute to overall animal 968 performance and health. Future studies should continue to address the influence of 969 dietary protein composition on peptide and AA transporter expression in the gut. Many of 970 the studies reported to this point include mRNA abundance data and it is unclear if 971 protein expression and activity parallel changes in the level of the transcript. As we gain a 972 better understanding of how AA assimilation may be improved through dietary peptides 973 we should strive for improved technology that allows for the accurate determination of 974 peptide profiles in order to better fine-tune these protein sources to address the nutritional 975 requirements of the animal. 43 Downloaded from jas.fass.org by on April 3, 2010. 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Distribution of 1510 the H+/peptide transporter PepT1 in human intestine: up-regulated expression in 1511 the colonic mucosa of patients with short-bowel syndrome. Am. J. Clin. Nutr. 75: 1512 922-930. 1513 Zimmerman, D. R., J. C. Sparks, and C. M. Cain. 1997. Carry-over responses to an 1514 intestinal hydrolysate in weanling pig diets. J. Anim. Sci. 75 (Suppl. 1): 71 1515 (Abstr.). 67 Downloaded from jas.fass.org by on April 3, 2010. Journal of Animal Science Page 68 of 74 1516 Table 1 The Proton Oligopeptide Cotransporter family 1517 (Based on Daniel and Kottra, 2004) Human Protein Aliases Substrates Transport Tissue gene name type/coupling distribution/cellular ion expression SLC15A1 PEPT1 Oligopeptide Di-, and Cotransport, Intestine and transporter 1, tripeptides H+ kidney apical H+/peptide protons membrane, transporter 1 lysosomal membrane SLC15A2 PEPT2 Oligopeptide Di-, and Cotransport, Kidney, lung, brain, transporter 2, tripeptides H+ mammary gland, H+/peptide protons bronchial transporter 2 epithelium SLC15A3 hPTR3 Peptide/ Histidine, Cotransport, Lung, spleen, histidine di- and H+ thymus, brain, liver, transporter 2, tripeptides adrenal gland, heart human protons peptide transporter 3, PHT2 SLC15A4 PTR4 Peptide/ Histidine, Cotransport, Brain, retina, histidine di- and H+ placenta transporter 1, tripeptides human protons peptide transporter 4, PHT1 1518 68 Downloaded from jas.fass.org by on April 3, 2010. Page 69 of 74 Journal of Animal Science 1519 Table 2. Comparison of cloned intestinal peptide transporter, PepT1, across several 1520 species. Species cDNA1 AA2 GenBank accession3 Source Rabbit 2,746 707 U06467 (NM_001082337) Fei et al., 1994; Boll et al., 1994 Sheep 2,829 707 AY027496 (NM_001009758) Pan et al., 2001 Cattle 2,742 707 BC140526 (NM_001099378) n/a Dog 3,026 708 AF461733 (NM_001003036) n/a Human 3,104 708 U21936 (NM_005073) Liang et al., 1995 Pig 2,698 708 AY180903 (NM_214347) Klang et al., 2005 Cynomolgus monkey 2,127 708 AY289936 Zhang et al., 2004 Rhesus monkey 3,108 708 AY289934 Zhang et al., 2004 Mouse 3,128 709 AF205540 (NM_053079) Fei et al., 2000 Rat 3,032 710 D50664 (NM_057121) Saito et al., 1995; Miyamoto et al., 1996 Chicken 2,914 714 AY029615 (NM_204365) Chen et al., 2002b Turkey 2,921 714 AY157977 Van et al., 2005 Zebrafish 2,636 718 AY300011 Verri et al., 2003 Atlantic cod 2,838 729 AY921634 Ronnestad et al., 2007 1 1521 The cDNA size reported as number of nucleotides. For the cynomolgus monkey, only 1522 the open reading frame is reported in GenBank. For the pig, two PepT1 cDNA were 1523 isolated, which contain alternative polyadenylation sites. On northern blots, two mRNA 1524 of 2.9 and 3.5 kb were detected in pig small intestine (Klang et al., 2005) and two mRNA 1525 of 3.4 and 4.0 kb were detected in monkey small intestine (Zhang et al., 2004). 2 1526 The protein size reported as number of AA. 3 1527 The source sequence ID is listed for each PepT1 gene entry and where applicable the 1528 NCBI reference ID is followed in parentheses. 69 Downloaded from jas.fass.org by on April 3, 2010. Journal of Animal Science Page 70 of 74 1529 Table 3. Comparison of IC50 and Kt values for various di-, tri-, and tetrapeptides among 1530 sheep, chicken, pig and human (no Kt values available for pig and human)1. Peptide Sheep Sheep Chicken Chicken Pig Human (Kt)2 (IC50)3 (Kt)4 (IC50)5 (IC50)6 (IC50)7 Arg-Lys 1.4 8.1 Glu-Glu 0.029 0.62 Gly-Met 0.10 0.05 0.03 Gly-Sar 0.61 0.47 0.94 1.2 Leu-Trp 0.06 0.02 Leu-Val 0.16 0.06 Lys-Lys 0.07 0.74 6.3 7.9 3.8 10.9 Lys-Met 0.051 0.16 0.11 0.04 Lys-Phe 0.024 0.15 0.11 0.03 Lys-Trp 0.03 0.66 Met-Glu 0.048 0.037 0.06 0.02 0.53 Met-Gly 0.18 0.016 0.08 0.27 0.005 Met-Leu 0.073 0.021 0.13 0.04 0.004 Met-Lys 0.45 0.12 0.17 0.07 Met-Met 0.027 0.024 0.08 0.02 0.013 Phe-Phe 0.019 0.08 Trp-Ala 0.07 0.04 0.26 Trp-Gly 0.06 0.06 0.73 Trp-Leu 0.12 0.02 Trp-Phe 0.03 0.02 0.06 Val-Leu 0.21 Leu-Gly-Gly 0.65 0.13 0.25 0.08 Leu-Ser-Phe 0.071 Lys-Trp-Lys 3.0 3.7 6.9 5.9 2.2 Lys-Tyr-Lys 2.0 Met-Leu-Phe 0.15 0.014 0.55 0.04 0.4 Leu-Gly-Gly 0.27 Thr-Ser-Lys 0.68 3.0 Met-Gly-Met-Met NR* 0.95 NR* 3.9 NC* Pro-Phe-Gly-Lys NR* 2.1 NR* 4.3 NC* Val-Gly-Asp-Glu NR* NR* Val-Gly-Ser-Glu NR* 6.9 NR* 27.0 NC* 1531 *NR/NC : No Response/ Non Calculatable 1 1532 All values expressed as mM 2 1533 Pan et al., 2001; injection into Xenopus oocytes 70 Downloaded from jas.fass.org by on April 3, 2010. Page 71 of 74 Journal of Animal Science 3 1534 Chen et al., 2002a; transfection in CHO cells 4 1535 Chen et al., 2002a; injection into Xenopus oocytes 5 1536 Chen et al., 2002b; transfection in CHO cells 6 1537 Klang et al, 2005; transient transfection in CHO cells 7 1538 Vig et al., 2006; transfection in MDCK cells 71 Downloaded from jas.fass.org by on April 3, 2010. Journal of Animal Science Page 72 of 74 1539 72 Downloaded from jas.fass.org by on April 3, 2010. Page 73 of 74 Journal of Animal Science 1540 Manuscript ID E-2007-0826 entitled, “Peptide Absorption and Utilization: Implications 1541 for Animal Nutrition and Health” 1542 Figure 1. Potential routes of peptide uptake in enterocytes. (A) The primary route of di- 1543 and tripeptide absorption is through cotransport with H+ by the peptide transporter, 1544 PepT1. (B) Cell-penetrating peptides (CPP) are capable of carrying cargo such as 1545 peptides to the inside of cells. (C) Increased permeability of tight junctions permits 1546 uptake of peptides via the paracellular route. 73 Downloaded from jas.fass.org by on April 3, 2010. Journal of Animal Science Page 74 of 74 Potential routes of peptide uptake in enterocytes. (A) The primary route of di- and tripeptide absorption is through cotransport with H+ by the peptide transporter, PepT1. (B) Cell-penetrating peptides (CPP) are capable of carrying cargo such as peptides to the inside of cells. (C) Increased permeability of tight junctions permits uptake of peptides via the paracellular route. 190x121mm (600 x 600 DPI) Downloaded from jas.fass.org by on April 3, 2010. Citations This article has been cited by 2 HighWire-hosted articles: http://jas.fass.org#otherarticles Downloaded from jas.fass.org by on April 3, 2010.
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