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									  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;




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                                          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: webbk@vt.edu




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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


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          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



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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



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          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



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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



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         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




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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




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         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.



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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



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         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-




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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



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         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




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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.




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         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



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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




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         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




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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




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         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




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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




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         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



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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




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         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



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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,




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         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.




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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




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         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




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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.




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         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




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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.



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         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



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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




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         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




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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




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         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.,



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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




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         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,



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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




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         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



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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




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         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.




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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



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         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.




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976                                  LITERATURE CITED

977   Adibi, S. A. 1971. Intestinal transport of dipeptides in man: Relative importance of

978          hydrolysis and intact absorption. J. Clin. Invest. 50:2266-2275.

979   Adibi, S. A. 1986. Kinetics and characteristics of absorption from an equimolar mixture

980          of 12 glycyl-dipeptides in human jejunum. Gastroenterology. 90:577-582.

981   Adibi, S. A. 1997. The oligopeptide transporter (Pept-1) in human intestine: biology and

982          function. Gastroenterology. 113:332-340.

983   Adibi, S. A. 2003. Regulation of expression of the intestinal oligopeptide transporter

984          (PepT-1) in health and disease. Am. J. Physiol. Gastrointest. Liver Physiol. 285:

985          G779-G788.

986   Adibi, S. A. and E. Phillips. 1968. Evidence for greater absorption of amino acids from

987          peptide than from free form in human intestine. Clin. Res. 16:446-448.

988   Adibi, S. A., G. A. Paleos, and E. L. Morse. 1986. Influence of molecular structure on

989          half-life and hydrolysis of dipeptides in plasma: importance of glycine as N-

990          terminal amino acid residue. Metabolism. 35:830-836.

991   Addison, J. M., D. M. Matthews and D. Burston. 1974. Competition between carnosine

992          and other peptides for transport by hamster jejunum in vitro. Clin. Sci. Mol. Med.

993          46:707-714.

994   Albritton L. M., L. Tseng., D. Scadden, and J. M. Cunningham. 1989. A putative murine

995          ecotropic retrovirus receptor gene encodes a multiple membrane-spanning protein

996          and confers susceptibility to virus infection. Cell. 57:659–666.

997   Amasheh, S., U. Wenzel, M. Boll, D. Dorn, W. Weber, W. Clauss, and H. Daniel. 1997.

998          Transport of charged dipeptides by the intestinal H+/peptide symporter PepT1



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         999           expressed in Xenopus laevis oocytes. J. Membr. Biol. 155:247-256.

        1000    Ashida, K., T. Katsura, H. Motohashi, H. Saito, and K.-I. Inui. 2002. Thyroid hormone

        1001           regulates the activity and expression of the peptide transporter PepT1 in Caco-2

        1002           cells. Am. J. Physiol. Gastrointest. Liver Physiol. 282:G617-G623.

        1003    Barbot, L., E. Windsor, S. Rome, V. Tricottet, M. Reynes, A. Topouchian, J. Huneau, J.

        1004           Gobert, D. Tome, and N. Kapel. 2003. Intestinal peptide transporter PepT1 is

        1005           over-expressed during acute cryptosporidiosis in suckling rats as a result of both

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        1290    Nielsen, C., J. Amstrup, R. Nielsen, B. Steffansen, S. Frokjaer, and B. Brodin. 2003.

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        1298           Sci. 39:446-451.




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1299

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        1515           (Abstr.).




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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




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        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.




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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



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                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




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            Journal of Animal Science                      Page 72 of 74




1539




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        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.




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                                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)




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