REVIEW ARTICLE

Transport of di- and tripeptides in teleost fish intestine

TizianoVerri1, Alessandro Romano1, Amilcare Barca1, Gabor Kottra2, Hannelore Daniel2 &Carlo Storelli1

1Laboratory of General Physiology, Department of Biological and Environmental Sciences andTechnologies, University of

Salento (formerly University of Lecce), Lecce, Italy2Molecular Nutrition Unit, Nutrition and Food Research Center, Technical University of Munich, Freising-Weihenstephan,

Germany

Correspondence:T.Verri, Laboratory of General Physiology, Department of Biological and Environmental Sciences and Technologies,

University of Salento (formerly University of Lecce),Via Provinciale Lecce-Monteroni, I-73100 Lecce, Italy. E-mail: tiziano.verri@unile.it

Abstract

The initial observation of peptide absorption in ¢shintestine dates back to 1981, when, in rainbow trout(Oncorhynchus mykiss), the rate of intestinal absorp-tion of the dipeptide glycylglycine (Gly-Gly) was com-pared in vivo with the rate of absorption of itscomponent amino acid glycine (Gly). The descriptionof the identi¢cation of the underlying mechanismsthat allow di- and tripeptide transport across theplasma membranes in ¢sh was provided in 1991,when the ¢rst evidence of peptide transport activitywas reported in brush-border membrane vesicles ofintestinal epithelial cells of Mozambique tilapia(Oreochromis mossambicus) by monitoring uptakeof radiolabelled glycyl-L-phenylalanine (Gly-L-Phe).Since then, the existence of a carrier-mediated, H1-dependent transport of di- and tripeptides (H1/pep-tide cotransport) in the brush-border membrane of¢sh enterocytes has been con¢rmed in many teleostspecies by a variety of biochemical approaches, pro-viding basic kinetics and substrate speci¢cities ofthe transport activity. In 2003, the ¢rst peptide trans-porter from a teleost ¢sh, i.e. the zebra¢sh (Danio re-rio) PEPtide transporter 1 (PEPT1), was cloned andfunctionally characterized in the Xenopus laevis oo-cyte expression system as a low-a⁄nity/high-capa-city system. PEPT1 is the protein in brush-bordermembranes responsible for translocation of intactdi- and tripeptides released from dietary protein byluminal and membrane-bound proteases and pepti-dases. The transporter possesses a⁄nities for thepeptide substrates in the 0.1^10mM range, depend-

ing on the structure and physicochemical nature ofthe substrates. After the molecular and functionalcharacterization of the zebra¢sh transporter, the in-terest in PEPT1 in teleost ¢sh has increased and ap-proaches for cloning and functional characteriza-tion of PEPT1 orthologues from other ¢sh species,some of them of the highest commercial value, arenow underway. In this paper, we provide a brief over-view of the transport of di- and tripeptides in teleost¢sh intestine by recalling the bulk of biochemical,biophysical and physiological observations collectedin the pre-cloning era and by recapitulating the morerecent molecular and functional data.

Keywords: gut, protein digestion, peptide absorp-tion, peptide transport, di- and tripeptides, PEPtidetransporter 1 (PEPT1)/SoLute Carrier 15 familymemberA1 (SLC15A1)

Introduction

Hydrolysis of dietary proteins leads to high levels ofshort-chain peptides (di- and tripeptides) in the in-testinal lumen during the digestive processes. Thedi- and tripeptides released are either further hydro-lysed to their constituent amino acids or directly ta-ken up in intact form into intestinal epithelial cells.Following apical in£ux, di- and tripeptides are se-quentially hydrolysed by multiple cytosolic hydro-lases (di- and tripeptidases), followed by basolaterale¥ux of the resulting amino acids via di¡erent aminoacid-transporting systems. Peptides not undergoing

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hydrolysis can exit the cell by a basolateral peptide-transporting system, not yet identi¢ed on a molecu-lar basis, and/or by other basolateral solute transportsystems that have occasionally been shown to allowtransport of selected peptides (for a comprehensivereview, see e.g. Daniel 2004).At the apical membrane of enterocytes, transport

of di- and tripeptides is mediated by a single carriersystem, called PEPT1 (PEPtide transporter 1) orSLC15A1 (SoLute Carrier15 family memberA1), afterclassi¢cation of membrane transporters by the Hu-man Genome Organization Nomenclature Commit-tee (for a recent review of the transporters of theSLC15 family, see e.g. Daniel & Kottra 2004). PEPT1functions as an Na1-independent, H1-dependenttransporter for a large variety of di- and tripeptides.Neither free amino acids nor peptides containingfour or more amino acids are accepted as substrates.The transport of di- and tripeptides is electrogenicand responds to both an inwardly directed trans-membrane H1 gradient (pHoutopHin) and (internalnegative) a transmembrane electrical potential (fordetails, see Daniel 2004). Transport is enantio-selec-tive and involves a variable proton-to-substrate stoi-chiometry for uptake of neutral and mono- orpolyvalently charged peptides. PEPT1 is also respon-sible for the transport of orally active drugs, such asb-lactam antibiotics, aminopeptidase and angioten-sin-converting enzyme (ACE) inhibitors, d-aminole-vulinic acid and many selected pro-drugs (for areview, see e.g. Rubio-Aliaga & Daniel 2002),although the option that ACE inhibitors may simplyinteract without being transported has recently beenput forward (Knˇtter,Wollesky, Kottra, Hahn, Fischer,Zebisch, Neubert, Daniel & Brandsch 2008).

Transport of di- and tripeptides in teleostfish intestine: the pre-cloning era

Transport at the luminal barrier (brush-border membrane) of ¢sh enterocytes

Peptide transport in ¢sh has been described in detailin brush-border membranes of European eel (Anguillaanguilla) intestine (Verri, Ma⁄a & Storelli1992; Ma⁄a,Verri, Danieli,Thamotharan, Pastore, Ahearn & Stor-elli 1997; Verri, Ma⁄a, Danieli, Herget,Wenzel, Daniel& Storelli 2000;Verri, Danieli, Bakke, Romano, Barca,R�nnestad, Ma⁄a & Storelli 2008), Mozambique tila-pia (Oreochromis mossambicus) intestine (Reshkin &Ahearn 1991; Thamotharan, Gomme, Zonno, Ma⁄a,Storelli & Ahearn1996), copper rock¢sh (Sebastes caur-

inus) intestine and pyloric ceca (Thamotharan &Gomme et al. 1996), Atlantic salmon (Salmo salar) in-testine and pyloric ceca (Bakke-McKellep, Nordrum,Krogdahl & Buddington 2000; Nordrum, Bakke-McKellep, Krogdahl & Buddington 2000), rainbowtrout (Oncorhynchus mykiss) intestine and pyloric ceca(Boge¤ , Rigal & Peres 1981; Buddington & Diamond1986,1987; Nordrum et al. 2000) and Antarctic croco-dile ice¢sh (Chionodraco hamatus) intestine (Ma⁄a,Rizzello, Acierno,Verri, Rollo, Danieli, D˛ring, Daniel& Storelli 2003). Interestingly, some of these early stu-dies pointed out that in ¢sh, as in mammals (for a re-cent review, see e.g. Daniel 2004), amino acids inpeptide-bound form can be absorbed more e⁄cientlythan themixture of the constituent amino acids (Boge¤et al.1981; Reshkin & Ahearn1991).In ¢sh brush-border membrane vesicle (BBMV) pre-

parations [a BBMV preparation is a highly puri¢edfraction of brush-border membranes mostly consist-ing (up to 80%) of closed and right side out orientedvesicles. Brush-border membrane vesicles are suitablefor a variety of in vitro studies, among which trans-membrane transport experiments (for details, see e.g.Biber, Stieger, Stange & Murer 2007, and literature ci-ted therein)], carrier-mediated uptake of radiolabelledpeptides is a saturable process withMichaelis^Mentenkinetics, and is stimulated by a transmembrane elec-trical potential and to a lesser extent by an inwardlydirected transmembrane H1 gradient (Reshkin &Ahearn1991;Thamotharan & Gomme et al.1996; Maf-¢a et al.1997;Verri et al. 2000). Interestingly, in BBMV,intravesicular acidi¢cation is observed with the addi-tion of di- and tripeptides to the extravesicular med-ium (Verri et al. 1992, 2000; Ma⁄a et al. 1997, 2003).Furthermore, due to the electrogenic nature of thetransport ^ e.g. the transport of the peptide is coupledto the transfer of positive charge(s) from the externalto the internal side of the membrane ^ addition of di-and tripeptides to the extravesicular medium inducessigni¢cant membrane potential depolarization (Verriet al. 2008). Diethylpyrocarbonate (DEP), which e⁄-ciently inhibits peptide transport in mammalianBBMV (Miyamoto, Ganapathy & Leibach 1986; Kra-mer, Girbig, Petzoldt & Leipe 1988; Kato, Maegawa,Okano, Inui & Hori 1989), also inhibits peptide trans-port in ¢sh BBMV (Verri et al. 1992, 2000, 2008; Tha-motharan & Gomme et al. 1996; Ma⁄a et al. 1997,2003).Taken together, the BBMVdata provide a strongbiochemical basis for the understanding of the car-rier-mediated mechanism that allows di- and tripep-tide uptake across the apical barrier of teleost ¢shenterocytes, and establish that a single carrier system

Di- and tripeptide transport in ¢sh gut T.Verri et al. Aquaculture Research, 2010, 41, 641^653

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with apparent Km values (Km,app) (Km,app indicates theapparent concentration of di- or tripeptide that yieldsone-half of maximal in£ux) in the 0.1^10mM rangemediates the transport process.This system transportsa large variety of di- and tripeptides, with remarkabledi¡erences in substrate a⁄nity and maximal velocitydepending on the particular amino acid compositionof the peptide substrate used for assay. As in mamma-lian systems, the transport is stereoselective, with apreference for L-a amino acids, although peptides con-taining D-isomers of amino acids (such as D-Phe-L-Ala)are also accepted (Verri et al. 2000; Ma⁄a et al. 2003),their a⁄nity for interaction largely depending on thelocation of the D-amino acid residue in the peptidebackbone and the polarity of the amino acidside chain (see e.g. Daniel 2004). As assessed by cis-in-hibition experiments, two di¡erent peptides that are si-multaneously present in the extravesicular spacecompete for the same transport system (Reshkin &Ahearn1991;Thamotharan & Gomme et al.1996; Maf-¢a et al.1997;Verri et al. 2000).The same holds true forthe presence of a peptide and a peptide-like drug (suchas the aminocephalosporin antibiotic cephalexin;Ma⁄a et al.1997). As a general observation, in kineticexperiments inwhich BBMVs are used in conjunctionwith a radioactive peptide, a linear apparent di¡usionprocess having a rate that is proportional to the extra-vesicular peptide concentration is always evidentbesides the saturable (Michaelis^Menten type)carrier-mediated component (Reshkin & Ahearn1991; Thamotharan & Gomme et al. 1996; Ma⁄a et al.1997; Verri et al. 2000). This di¡usive pathway, whichmay include a carrier-independent in£ux and/or very

low-a⁄nity, high-capacity carrier systems (as sug-gested by Thamotharan & Gomme et al. 1996), ac-counts for a signi¢cant fraction of the peptide uptakein teleost ¢sh BBMV.The rates of dipeptide absorption by ¢sh intestine

have also been measured using intact tissue of Atlan-tic salmon and rainbow trout (Bakke-McKellep et al.2000; Nordrum et al. 2000) in conjunction with theeverted sleevemethod (Karasov & Diamond1983; Bud-dington, Chen & Diamond1987). (By mounting the in-testinal sleeve on a grooved rod, this method allowsisolation of the serosal surface from the incubationmedium, so that one measures only uptake across theapical membrane rather than across both cell faces si-multaneously. In addition, sleeves from di¡erent seg-ments of the alimentary canal can be mounted ondi¡erent rods, thus allowing regional analysis of nutri-ent uptake.) In these experimental setups, in bothAtlantic salmon and rainbow trout peptide transportacross the brush-border membrane barrier appearsas a combinationof carrier-mediated andapparent dif-fusion processes, the latter probably accounting forthe majority of total uptake at higher concentrationsof peptides (Bakke-McKellep et al. 2000; Nordrumet al. 2000). In salmonids, there is a declining proxi-mal-to-distal gradient of peptide absorption along thepost-gastric intestinal tract (pyloric ceca4proximalintestine4mid-intestine4distal intestine), which issimilar to that found for absorption of both glucoseand amino acids (Buddington & Diamond 1986, 1987;Bakke-McKellep et al. 2000; Nordrum et al. 2000).Furthermore, in each region, the rates of peptide up-take fall within the same order of magnitude as those

L-Ala

L-Asp Gly

L-His

L-Le

uL-

Lys

L-M

et

L-Phe

L-Val

Carno

sine

0.0

0.5

1.0

1.5

2.0

2.5

3.0 Pyloric ceca

Proximal intestine

Mid intestine

Distal intestine

Nut

rient

upt

ake,

Vm

ax(n

mol

min

–1 m

g tis

sue–1

)

Figure 1 Uptakes of nine amino acids and the dipeptide b-Ala-L-His (carnosine) (25mM) in rainbow trout gut. Uptakerates are normalized to tissue weight (adapted from Buddington & Diamond1987). Nutrient concentrations of 25mMareadequate to saturate the carriers and yield the maximal rate of active nutrient uptake (Vmax).Values are means � SEM offour ¢sh.

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of amino acid uptake (Fig.1). The basic features of thecarrier-mediated peptide transport process occurringat the brush-border membrane of teleost ¢sh absorb-ing intestinal cells are summarized in Table 1 and inthe literature cited therein.

Transport at the contraluminal barrier(basolateral membrane) of ¢sh enterocytes

To our knowledge, only one paper has focused on thepathway for peptide transport across the basolateralmembrane of teleost ¢sh enterocytes. Using basolat-eral membrane (BLMV) preparations in conjunctionwith the radioactive tracer [14C]Gly-Sar,Thamothar-an, Zonno, Storelli and Ahearn (1996) have detectedGly-Sar transport at the basolateral membrane ofMozambique tilapia enterocytes. Kinetic analysis ofthe basolateral transport rate revealed that the trans-port occurs by a saturable process conforming to Mi-chaelis^Menten kinetics, with a Km,app for Gly-Sar of13.3 � 3.8mM. This transport activity is almost in-sensitive to DEP. Furthermore, [14C]Gly-Sar in£uxinto tilapia BLMV shows cis-inhibition by severalother dipeptides.In summary, transcellular transport of peptides

across teleost ¢sh post-gastric intestinal barrier canbe accounted for bya combination of carrier-mediatedand apparent di¡usion routes. At the brush-bordermembrane level, peptide transport occurs by a well-characterized carrier-mediated process in conjunc-tion with an apparently di¡usive process, which mayinclude (a) carrier-independent in£ux and/or putativevery low-a⁄nity, high-capacity carrier system(s). Ab-sorbed di- and tripeptides may be hydrolysed to com-ponent amino acids within the cell before basolateralexit via amino acid transport systems or leave the cellintact via a not yet well characterized basolateraltransporter. In intact tissue, paracellular transport ofdi- and tripeptides cannot be excluded.

Transport of di- and tripeptides in teleostfish intestine: the cloning era

The zebra¢sh (Danio rerio) PEPT1 transporterparadigm

In the last decades, the cyprinid D. rerio (zebra¢sh), asmall (3^4 cm long) freshwater teleost ¢sh, hasemerged as a powerful model organism for experi-mental biology, vertebrate embryology, developmen-tal genetics and toxicology studies. In addition, its

importance as an animal model for regulatory andintegrative physiology studies has steadily increased(for a review, see e.g. Briggs 2002). In 2003, we suc-ceeded in the molecular cloning of zebra¢sh PEPT1,the ¢rst PEPT1-type peptide transporter from a tele-ost ¢sh (Verri, Kottra, Romano, Tiso, Peric, Ma⁄a,Boll, Argenton, Daniel & Storelli 2003). Zebra¢shPEPT1 complementary DNA (cDNA) (zfPEPT1; Gen-Bank Acc. No. AY300011) is 2746 bp long, with anopen reading frame of 2157 bp encoding a putativeprotein of 718 amino acids. Hydropathy analysis pre-dicts at least 12 potential membrane-spanning do-mains (TMDs) with a large extracellular loopbetween TMDs IX and X. The predicted zebra¢shPEPT1amino acid sequence exhibits a percentage ofidentity of 60.3^61.5% when compared with otherPEPT1-type members of the SLC15 family alreadycharacterized from mammals and birds.As assessed by reverse transcription-polymerase

chain reaction (RT-PCR), zebra¢sh PEPT1 mRNA ishighly expressed in the intestine of adult ¢sh, as wellas in the kidney and spleen, although to a lower ex-tent. During larval development, PEPT1 mRNA ex-pression starts by 2 days post fertilization (dpf),slightly increases by 3 dpf and reaches the highestobserved values by 4^7 dpf. In this time frame, ex-pression is strictly localized at the proximal intestinallevel (i.e. the intestinal bulb; for a recent descriptionof the digestive system development in zebra¢sh, seee.g. Ng, de Jong-Curtain, Mawdsley,White, Shin, Ap-pel, Dong, Stainier & Heath 2005), as assessed bywhole-mount in situ hybridization analysis. NomRNA signal is detectable in the mid- and posteriorintestine or other tissues/organs of the developingembryo (Verri et al. 2003).To characterize zebra¢sh PEPT1 function, the

complementary RNA (cRNA) originating fromzfPEPT1has been injected intoXenopus laevis oocytesto allow biosynthesis and expression of the func-tional protein at the oocyte plasma membrane. [Thisexperimental manoeuvre allows biosynthesis offunctional heterologous proteins by the Xenopus lae-vis oocyte ‘expression system’, which is able to e⁄-ciently transcribe and translate injected geneticinformation, perform assembly of the foreign proteinproducts, correctly process the nascent polypeptidesand target them to the proper subcellular compart-ment of the oocyte (i.e. the plasma membrane in thecase of plasma membrane proteins; for a comprehen-sive review, see e.g. Romero, Kanai, Gunshin & Hedi-ger 1998).] To establish the basic kinetic properties ofthe zebra¢sh PEPT1 transporter, the zwitterionic

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Table 1 Characteristics of carrier-mediated peptide transport in teleost ¢sh post-gastric alimentarycanal (intestinal) regions

Species

Develop-mentalstage Tissue Method

Testsubstrate

Km,app

(mM) References

European eel�

(Anguilla anguilla)

Yellow eel Whole intestine a Gly-L-Pro 1.27 � 0.01w,z,‰ Maffia et al. (1997)

b 1.32 � 0.10w,z,‰ Maffia et al. (1997)

b 1.04 � 0.31w,z,‰ Verri et al. (2000)

c 1.43 � 0.53w,z,‰ Verri et al. (2008)

c 1.68 � 1.01z,z,‰ Verri et al. (2008)

a D-Phe-L-Ala 0.74 � 0.16w,z,‰ Verri et al. (2000)

b 1.19 � 0.52w,z,‰ Verri et al. (2000)

b Gly-Gly 12.36 � 3.13w,z,‰ Verri et al. (1992)

b 1.81 � 0.49w,z,‰ Verri et al. (2000)

c 1.59 � 0.40w,z,‰ Verri et al. (2008)

c 2.49 � 0.84z,z,‰ Verri et al. (2008)

b Gly-L-Ala 0.97 � 0.42w,z,‰ Verri et al. (2000)

b Gly-L-Asn 2.59 � 0.73w,z,‰ Verri et al. (2000)

b Gly-Sar 1.75 � 0.47w,z,‰ Verri et al. (2000)

b L-Pro-Gly 0.87 � 0.36w,z,‰ Verri et al. (2000)

Mozambique

tilapiak(Oreochromis

mossambicus)

Sexually

immature

adult

Whole intestine a Gly-L-Phe 9.8 � 3.5w,��,‰ Reshkin and Ahearn

(1991)

Upper one-half of

the intestine

a Gly-Sar 0.56 � 0.08w,��,‰ Thamotharan,

Gomme et al. (1996)

Antarctic crocodile

icefish�

(Chionodraco

hamatus)

Sexually

mature

adult

Whole intestine b Gly-L-Pro 0.806 � 0.161w,ww Maffia et al. (2003)

Atlantic salmon�

(Salmo salar)

Smolt Pyloric caecazz d Gly-L-Pro 0.5 � 0.4z,��,‰‰ Bakke-McKellep et al.

(2000)

Mid-intestinezz d 1.5 � 0.4z,��,‰‰ Bakke-McKellep et al.

(2000)

Distal intestinezz d NDz,��,‰‰ Bakke-McKellep et al.

(2000)

Proximal intestinezz d Gly-Sar 8.579 � 5.327z,z,‰‰ Nordrum et al. (2000)

13.120 � 6.620z,��,‰‰ Nordrum et al. (2000)

1.370 � 0.118z,��,‰‰,zz Nordrum et al. (2000)

Rainbow trout�

(Oncorhynchus

mykiss)

Smolt Proximal intestinezz d Gly-Sar 9.774 � 8.736z,z,‰‰ Nordrum et al. (2000)

0.747 � 0.051z,��,‰‰ Nordrum et al. (2000)

5.270 � 1.41z,��,‰‰,zz Nordrum et al. (2000)

Km,app indicates the apparent concentration of dipeptide that yielded one-half of maximal in£ux (Jmax). Km,app values are expressed inmM and are reported as means � SEM of n, number of observations (see references for details). Jmax data have not been reported in thistable as the use of di¡erent experimental approaches make them not comparable (see references for details). Methods: a, brush-bordermembrane vesicles (BBMV) in conjunction with a radioactive dipeptide to directly monitor dipeptide uptake; b, BBMV in conjunctionwith the pH-sensitive dye acridine orange to monitor dipeptide-dependent intravesicular acidi¢cation; c, BBMV in conjunction with thevoltage-sensitive dye 3,3 0-diethylthiacarbocyanine iodide [DiS-C2(5)] to monitor dipeptide-dependent membrane potential depolariza-tion; d, everted sleeves in conjunction with a radioactive dipeptide to directly monitor dipeptide uptake.�Carnivorous.wTransport measured in the absence of extravesicular (BBMV) or extracellular (everted sleeves) sodium.zSeawater-adapted ¢sh.‰Transport activity measured at 25 1C.zTransport measured in the presence of extravesicular (BBMV) or extracellular (everted sleeves) sodium.kOmnivorous (http://www.¢shbase.org).��Freshwater-adapted ¢sh.wwTransport activity measured at 0 1C.zzDivision of intestine according to Buddington and Diamond (1987).‰‰Transport activity measured at 10 1C.zzSoybean meal-fed ¢sh.ND, not detected.

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dipeptide Gly-L-Gln, a well-known high-a⁄nity sub-strate for mammalian PEPT1 transporters, has beenused as a test compound in two-electrode voltageclamp (TEVC) experiments (Verri et al. 2003), underthe same experimental conditions as for mammalianPEPT1 analysis (Kottra & Daniel 2001; Kottra et al.2002). Current^voltage (I^V) relationships (Fig. 2) in-dicate that zebra¢sh PEPT1, similar to the mamma-lian orthologues, transports electrogenically notonly in the forward but also in the reverse direction,thus suggesting not only in£ux but also e¥ux of pep-tides across the plasmamembrane, at least under cer-tain imposed experimental conditions. [For instance,at pH 7.5, the Gly-L-Gln-dependent transport currentis inwardly directed at negative membrane poten-tials. However, the direction of current reverses (i.e.it becomes outwardlydirected) at more positivemem-brane potentials (at �7 and 147mV when 1 and20mMGly-L-Gln are applied respectively; see Fig. 2).]Gly-L-Gln transport occurs according to Michaelis^Menten kinetics (in oocytes clamped at �60mVandperfused with an uptake solution at pH 7.5, thekinetic parameters for Gly-L-Gln are K0.5 51.44� 0.18mM and Imax 5200 � 24 nA) [K0.5 indicatesthe apparent concentration of dipeptide that yieldsone-half of maximal transport current (Imax)] and isstrongly a¡ected by both membrane potential (seeFig. 2, where the dependence on membrane potentialis evident, with currents steadily decreasing, passingfrom ^160mV to less negative potentials) and extra-cellular pH (Table 2). It is noteworthy that the appar-ent a⁄nity for Gly-L-Gln at pH 5.5 is higher than thatat pH 6.5, the apparent a⁄nity at pH 6.5 is higherthan that at pH 7.5 and these three a⁄nities are sig-ni¢cantlyhigher (at least10-fold) than thatmeasured

at pH 8.5 (Table 2). This behaviour is very similar tothat observed with the mammalian transporters(Kottra & Daniel 2001). On the other hand, varyingextracellular pH from5.5 to 8.5 results, unexpectedly,in a pronounced increase (at least fourfold) in themaximal transport current (Table 2). This increasein Imax as the external proton concentration de-creases is the ¢rst description of such a kinetic beha-viour in any of the PEPT1-type transporters studiedusing the electrophysiological approach. Thus, inspite of several similarities, a striking di¡erence ex-ists between the kinetic behaviour of zebra¢sh PEPT1and that of the other known mammalian peptidetransporters.Recently, the results obtained with Gly-L-Gln have

been corroborated usingother test peptides for analy-sis. Brie£y, zebra¢sh PEPT1 translocates not onlyneutral (Gly-L-Gln) but also acidic (Gly-L-Asp, L-Asp-Gly) and basic (Gly-L-Lys, L-Lys-Gly) dipeptides usingan electrogenic process that follows Michaelis^Men-ten kinetics (similar to that described for Gly-L-Gln;seeTable 2). In particular, by comparing pairs of sub-strates with the same charged amino acid residues ineither the amino- or the carboxyl-terminal position

–200 –160 –120 –80 –40 40 80 120

–800

–600

–400

–200

200

Membrane potential, V (mV)

Cur

rent

, I (

nA)1 mM

20 mM

Figure 2 Steady-state current^voltage (I^V) relation-ships for Gly-L-Gln (1 and 20mM, pH 7.5) as measured inzebra¢sh PEPT1-expressing Xenopus laevis oocytes.Valuesare means � SEM of10 oocytes.

Table 2 Kinetic parameters of di¡erently charged dipep-tides (neutral, basic and acidic dipeptides) and their pHdependency

Substrate pHK0.5 (mM,at ^60mV)

Imax (relative toGly-Gln at pH 7.5)

No. ofoocytes(n)

Gly-L-Gln 8.5 9.6 � 1.6 1.62 � 0.10 10

7.5 1.44 � 0.18 1.00 ( 5 200 � 24 nA) 16

6.5 0.24 � 0.07 0.43 � 0.03 10

5.5 0.13 � 0.03 0.36 � 0.03 16

Gly-L-Lys 8.5 38 � 8 1.91 � 0.21 9

7.5 6.0 � 1.5 0.94 � 0.07 17

L-Lys-Gly 8.5 16 � 6 2.20 � 0.10 7

7.5 3.5 � 0.6 1.26 � 0.07 16

Gly-L-Asp 7.5 21 � 5 0.64 � 0.14 8

5.5 0.21 � 0.03 0.47 � 0.04 15

L-Asp-Gly 7.5 13 � 1 0.51 � 0.07 5

5.5 0.17 � 0.05 0.36 � 0.04 5

Electrophysiological data reported in this table were obtainedby experiments conducted on Xenopus laevis oocytes voltageclamped at ^60mV. Peptide-evoked inward currents were mea-sured in the presence of increasing concentrations of dipeptide(0.5^20mM) in oocytes perfused with solutions at various pHvalues (ranging from 5.5 to 8.5). K0.5 indicates the apparent con-centration of dipeptide that yielded one-half of maximal trans-port current (Imax). Values are reported as means � SEM of n,number of oocytes (see last column).The maximal transport current (Imax) values are relative to Imax

of Gly-L-Gln measured at pH 7.5 in the same oocyte.

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of the dipeptide (i.e. Gly-L-Asp vs. L-Asp-GlyandGly-L-Lys vs. L-Lys-Gly), it has been shown that the positionof the charged amino acid chain in a substrate signif-icantly a¡ects the transport process. Furthermore,usingTEVC experiments combinedwith intracellularpH recordings and voltage-clamped tracer measure-ments (Kottra & Daniel 2001; Kottra et al. 2002), thefollowing evidences have been provided: (a) directtransport of a peptide across the plasma membraneof X. laevis oocytes, by monitoring the uptake ofradiolabelled Gly-Sar ([14C]Gly-Sar); (b) direct evi-dence for dipeptide-mediated H1 translocation, bymonitoring zebra¢sh PEPT1-speci¢c intracellularacidi¢cation; and (c) preliminary £ux couplingstoichiometry, by comparing the initial rate of[14C]Gly-Sar uptake with the net charge £ux due toGly-Sar-induced inward current. In particular,the unexpected ¢nding of a proton-to-Gly-Sarcoupling coe⁄cient of 0.421 � 0.035 suggests thatH1/Gly-Sar transport via zebra¢sh PEPT1may occuraccording to a1:2 stoichiometric ratio (A. Romano &G. Kottra, unpubl. obs.).

Back to the role of PEPT1 in zebra¢shintestinal physiology

The zebra¢sh PEPT1-type peptide transporter oper-ates in the proximal intestine andmediates the trans-port of bulk quantities of di- and tripeptides arisingfrom protein digestion. Interestingly, transport ofboth neutral and charged peptides exhibits a highlypredictable, large increasewith increasing pH, whichis not found in any mammalian PEPT1-type peptidetransporters, and seems to represent a functionalhallmark of the zebra¢sh transporter. Also, expres-sion starts early (2 dpf) during embryo development.This may be placed in the context of the specialties ofzebra¢sh intestinal physiology.Zebra¢sh is a carnivorous ¢sh, which has its func-

tional correlate in the presence of a very short intest-inal tract. Moreover, zebra¢sh is a stomachless ¢sh(which implies that no acidic content is released intothe proximal intestine) and its intestinal lumenmight be alkaline under normal physiological condi-tions (due to pancreas and gallbladder secretions intothe intestinal lumen). That this holds true is sup-ported by the ¢nding of alkaline pH (47.5) in adultzebra¢sh intestinal lumen (Nalbant, Boehmer, Deh-melt,Wehner &Werner1999). Inmammals, the acidicmicroclimate layer occurring in the vicinity of the lu-minal cell surface of the small intestinal epithelium is

important for optimal absorption of peptides viaPEPT1 (see e.g. Daniel 2004; Thwaites & Anderson2007). This process depends on the trans-apical pHgradient that is maintained, at least in part, by theactivity of the brush-border membrane Na1/H1 ex-changer NHE3 (see e.g. Kennedy, Leibach, Ganapa-thy & Thwaites 2002; Thwaites, Kennedy, Raldua,Anderson, Mendoza, Bladen & Simmons 2002). Ithas been shown that NHE3 is not expressed in the in-testine of the Osorezan dace Tribolodon hakonensis(Hirata, Kaneko, Ono, Nakazato, Furukawa, Hasega-wa,Wakabayashi, Shigekawa, Chang, Romero & Hir-ose 2003), a cyprinid taxonomically related tozebra¢sh. Therefore, no acidi¢cation process (at leastvia NHE3) might occur in cyprinids and, as a conse-quence, no acidic microclimate pHmight be e¡ective,thus making the external cell surface of the intest-inal epithelium fully sense the luminal alkaline pH.[A similar functional arrangement might be e¡ectivein some other ¢sh species, such as the European eel,in which the limited stimulatory e¡ect on di- and tri-peptide transport exerted by an inwardly directedtransmembrane H1 gradient (see e.g. Ma⁄a et al.1997;Verri et al.2000) stronglycorrelates with the ab-sence of a brush-border membrane Na1/H1 exchan-ger (Vilella, Zonno, Lapadula,Verri & Storelli1995).] Inthis respect, the presence of a peptide transport sys-tem that is adapted to optimally operate at alkalinepH in the proximal part of the intestine would allowrapid and complete transport of peptides derivedfrom intestinal protein digestion. In particular, thepositive modulation by alkaline pH would be highlye¡ective as soon as the alkaline £uids from the pan-creas and gallbladder are released into the proximalintestinal lumen as a consequence of the presence offood in the proximal intestine, thus making the in-crease in the transport activity (see Table 2) func-tional to the rapid removal of the ingested andpartially digested dietary proteins.In the early zebra¢sh embryo, energy requirement

is provided by the yolk until full maturation of thealimentary canal occurs. The morphogenesis of thezebra¢sh digestive tract occurs by � 2.5 dpf, whencontiguous and histologically recognizable primor-dia of the pharynx, oesophagus and intestine are evi-dent. Then, further growth and di¡erentiation of alldigestive organ primordia occur so that by 5 dpf thedigestive system is functional and longitudinallydi¡erentiated in the mouth and oral cavity, pharynx,oesophagus, intestine, rectum and anus (see e.g.Wal-lace & Pack 2003; Ng et al. 2005; Wallace, Akhter,Smith, Lorent & Pack 2005). Full opening of the

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rostral digestive tract is achieved by � 3 dpf, whenthe lumen of the posterior pharinx is visible and themouth is open. The anus is open at 4 dpf. Epithelialpolarization occurs parallel to organ morphogenesis,with markers of apical membranes of the intestinalcells, e.g. b-actin and alkaline phosphatase, ¢rst de-tected at � 2.5 dpf and increasing from 3 (the endof the hatching period) to 4 dpf. By 4^5 dpf, the ante-rior intestine also undergoes a transition from astraight to a coiled tube and exhibits typical folding(see e.g.Wallace & Pack 2003). Zebra¢sh PEPT1 is al-ready expressed in the proximal intestine by 4 dpf,with strong signals in the intestinal bulb by 5 dpfand thereafter. It therefore appears that zebra¢shPEPT1 expression precedes the functional matura-tion of the gut, which occurs by 5 dpf and makes the¢sh ready to perform ¢rst feeding and digestion of ex-ternal food. By 5 dpf, the larva starts the active searchfor external food, although a remnant of the yolk isstill present. Taken together, these observations sup-port the notion that the intestinal bulb is the site ofthe most e⁄cient absorption of peptides in zebra¢sh,with PEPT1being expressed at high levels before theanimal starts to rely autonomously on external food.In summary, zebra¢sh PEPT1plays a pivotal role in

driving the absorption of dietary di- and tripeptides.Zebra¢sh PEPT1 is adapted to transport at alkalinepH. In the zebra¢sh, PEPT1 is expressed early in theproximal intestine (e.g. the intestinal bulb), startingat 2 dpf and thus preceding functional maturation ofthe gut, ¢rst feeding and complete yolk resorption.Currently, zebra¢sh PEPT1 is fully recognized as auseful marker to study zebra¢sh gut regionalization,di¡erentiation and morphogenesis (see e.g. Zecchin,Filippi, Biemar,Tiso, Pauls, Ellertsdottir, Gnˇgge, Bor-tolussi, Driever & Argenton 2003).

Towards the molecular and functionaldescription of PEPT1 in other ¢sh species

After the molecular and functional characterizationof the zebra¢sh transporter, the interest in PEPT1 inteleost ¢sh has grown. This increased attentionmainly comes from the observation that teleosts cane⁄ciently utilize dietary di- and tripeptides for devel-opment, growth and metabolism and, consequently,that balanced peptide-based diets or peptide ratherthan amino acid supplementation would be highlybene¢cial in solving the nutritional inadequacy offormulated feeds for cultured ¢sh (see e.g. Zamboni-no Infante, Cahu & Peres 1997; Dabrowski, Lee &

Rinchard 2003; Araga� o, Conceic� a� o, Martins, R�n-nestad, Gomes & Dinis 2004; Dabrowski, Terjesen,Zhang, Phang & Lee 2005; Tesser, Terjesen, Zhang,Portella & Dabrowski 2005;Terjesen, Lee, Zhang, Fail-la & Dabrowski 2006; Zhang, Dabrowski, Hliwa &Go-mulka 2006; R�nnestad, Kamisaka, Conceic� a� o,Morais & Tonheim 2007; Ostaszewska, Dabrowski,Hliwa, Gomo¤ zka & Kwasek 2008). This next sectionis intended to provide a brief overview of the currentstate of the art.PEPT1-related expressed nucleotide sequences as

found in various teleost ¢sh species and currentlyavailable in GenBank are summarized in Table 3.With a panel of ¢sh molecular tools available, severalcloning projects have been initiated and are now pro-gressing fast. The cloning of a cDNA encoding forAtlantic cod (Gadus morhua) PEPT1 has beenachieved (R�nnestad, Gavaia,Viegas,Verri, Romano,Nilsen, Jordal, Kamisaka & Cancela 2007) and, toour knowledge, there are at least six more cloningprojects underway, aimed at the functional charac-terization of PEPT1 in Antarctic crocodile ice¢sh(M. Ma⁄a, pers. comm.), Atlantic salmon (I. R�nnes-tad, pers. comm.), common carp (Cyprinus carpio; T.Ostaszewska & K. Dabrowski, pers. comm.), rainbowtrout (T. Ostaszewska & K. Dabrowski, pers. comm.),European sea bass (Dicentrarchus labrax; G. Terova &M. Saroglia, pers. comm.) and China rock¢sh (Se-bastes nebulosus; J. J. Amberg, pers. comm.). For twospecies, namelyAtlantic salmonandAntarctic croco-dile ice¢sh, the functional clones are already avail-able and the kinetic characterization in X. laevisoocytes is in progress (Table 3). Currently, the onlyfunctional data published in full paper form, besidesthe zebra¢sh, regard the preliminary functionalexpression of a H1/peptide cotransport activity afterinjection into X. laevis oocytes of total RNA isolatedfrom Antarctic crocodile ice¢sh intestinal mucosa.Injection of mRNA stimulates D-Phe-L-Ala uptake ina dose-dependent manner and an excess of Gly-L-Glnsigni¢cantly inhibits D-Phe-L-Ala transport (Ma⁄aet al. 2003).The rest of the published data on PEPT1-type pep-

tide transporters in ¢sh contribute towards under-standing the tissue distribution of PEPT1 in thespecies analysed, namely Atlantic cod, Orientalweatherloach (Misgurnus anguillicaudatus) and or-ange-spotted grouper (Epinephelus coioides).The Atlantic cod is a key commercial species in

many North Atlantic countries that has recentlybeen targeted for aquaculture, mainly due to deple-tion of natural stocks by over¢shing (see e.g. Brander

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2007). A full-length cDNAthat encodes for theAtlan-tic cod PEPT1-type peptide transporter has beencloned (R�nnestad, Gavaia et al. 2007). This cDNA(codPepT1; GenBank Acc. No. AY921634) is 2838 bplong, with an open reading frame of 2190 bp encod-ing a putative protein of 729 amino acids that shares63% identity with the zebra¢sh PEPT1. As for zebra-¢sh, hydropathy analysis suggests at least 12 poten-tial TMDs and a large extracellular loop betweenTMDs IX and X. Unfortunately, when injected into X.laevis oocytes, codPepT1 cRNA did not drive bio-synthesis of any functional protein (I. R�nnestad,pers. comm.). Therefore, no functional analysis of theAtlantic cod PEPT1is currentlyavailable (seeTable 3).In adult Atlantic cod, PEPT1 mRNA is highly ex-pressed in the intestine and well expressed in boththe kidneyand the spleen (avery slight RT-PCR signalis also detectable in the ovary; R�nnestad, Gavaiaet al. 2007). This expression pattern is similar to thatof zebra¢sh PEPT1. A more detailed analysis of theregional distribution along the intestinal tract indi-cates that Atlantic cod PEPT1 is ubiquitously ex-

pressed in all segments beyond the stomach,including the pyloric ceca (R�nnestad, Gavaia et al.2007. It exhibits a lower expression only in the mostposterior portion of the intestine. This ¢nding sug-gests that Atlantic cod may have a very high capacityto absorb small peptides from dietary protein diges-tion, with absorption occurring in most parts of thepost-gastric canal. The low expression in the last seg-ment that includes the hindgut indicates that thissegment is not, or only slightly, involved in peptideabsorption. In larval Atlantic cod, a detailed analysisof the regional distribution along the gut (based onquantitative RT-PCRand in situ hybridization studies)has revealed that PEPT1 mRNA is ubiquitously ex-pressed in the epithelium of all segments posteriorto the oesophagus, with the only exception of thesphincter regions (Amberg, Myr, Kamisaka, Jordal,Rust, Hardy, Koedijk & R�nnestad 2008). This spatialexpression pattern is observed at hatching and ismaintained in the more developed gut (22 dayspost hatching). Temporal analysis of expression inlarval Atlantic cod gut indicates that PEPT1 mRNA

Table 3 PEPtide transporter1 (PEPT1)-related expressed nucleotide sequences in various teleost ¢sh species as available inGenBank (annotated sequences only)

Description SpeciesGenBankAcc.No.

Functionalanalysis References

mRNA, complete cds Zebrafish (Danio rerio) AY300011 Yes Verri et al. (2003)

Antarctic crocodile icefish (Chionodraco

hamatus)

Not yet released Yes M. Maffia, pers. comm.

Atlantic salmon (Salmo salar) Not yet released Yes I. Rønnestad, pers. comm.

Atlantic cod (Gadus morhua) AY921634 No Rønnestad, Gavaia et al. (2007)

China rockfish (Sebastes nebulosus) EU160494 No GenBank submission�

European sea bass (Dicentrarchus labrax) FJ237043 No GenBank submissionw

mRNA, partial cds Antarctic crocodile icefish (Chionodraco

hamatus)

AY170828 – Maffia et al. (2003)

Oriental weatherloach (Misgurnus

anguillicaudatus)

DQ668370 – Goncalves et al. (2007)

Common carp (Cyprinus carpio) EU328390 – GenBank submissionzRainbow trout (Oncorhynchus mykiss) EU853718 – GenBank submission‰

European sea bass (Dicentrarchus labrax) AM419037 – GenBank submissionz

EST, expressed

sequence tags

Channel catfish (Ictalurus punctatus) CK416736 – GenBank submissionk

�Amberg J.J., Anderson C.L., Hill R.A., Rust M.B. & Hardy R.W. Aquaculture Research Institute, University of Idaho, Hagerman, ID, USA.wTerova G., Rimoldi S., Cora S., Bernardini G., Gornati R. & Saroglia M. Department of Biotechnology and Molecular Sciences, Univer-sity of Insubria,Varese, Italy.zNie G.X., Zheng J.L. & Song D.Y. Life Sciences, Henan Normal University, Xinxiang, Henan, China.‰Ostaszewska T., Szatkowska I., Muszynska M., Grochowski P. & Dabrowski K. Department of Ichtyobiology and Fisheries, WarsawAgricultural University,Warsaw, Poland.zHakimY. Animal Science, Hebrew University Jerusalem, Rehovot, Israel.kLiu Z.J. The Fish Molecular Genetics and Biotechnology Laboratory, Department of Fisheries and Allied Aquacultures and Program ofCell and Molecular Biosciences, Auburn University, Auburn, AL, USA.cds, coding sequence.

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is expressed before hatching, i.e. before the onset ofexogenous feeding. This pre-feeding expression ofthe transporter mRNA parallels the data obtainedwith zebra¢sh (Verri et al. 2003), chicken (Gallusgallus; Chen, Pan, Wong & Webb Jr 2005) and rat(Rattus norvegicus; Shen, Smith & Brosius III2001), thus suggesting a common theme amongvertebrate PEPT1 transporters. Expression increasesfollowing ¢rst-feeding and is set to high levels forat least 3 weeks (22 days post hatching; Amberget al. 2008).The Oriental weatherloach, a small freshwater co-

bitid teleost that lives in rice ¢elds, streams andditches, is a facultative air breather that makes useof its hindgut as an accessory air-breathing organ(McMahon & Burggren1987). It is agastric and has ashort straight gut. A 643 bp long cDNA (GenBankAcc. No. DQ668370) that encodes for a portion of anOriental weatherloach PEPT1-type peptide transpor-ter has been cloned (Gonc� alves, Castro, Pereira-Wil-son, Coimbra & Wilson 2007) and used to designprimers for tissue distribution analysis by RT-PCR.In Oriental weatherloach, PEPT1mRNA is highly ex-pressed in the gut andwell expressed in the heart andliver. A very slight signal is also detectable in thebrain. In contrast to tissue distribution analysis inzebra¢sh and Atlantic cod, no signal is evident in thekidney. In the gut, the highest expression is found inthe foregut and the midgut, which represent the di-gestive/absorptive zone of the intestinal tract. In thehindgut, which is the respiratory zone of the intes-tine, PEPT1expression is very low.One of the limits of expression studies in ¢sh is the

lack of adequate information at the protein level be-cause of the lack of species-speci¢c antibodies. Toour knowledge, the only protein expression dataavailable in ¢sh come from a recent study performedin the carnivorous orange-spotted grouper, using apolyclonal rabbit anti-PEPT1antibody raised againsta recombinant protein of human origin that wasshown to cross-react with orange-spotted grouperPEPT1 protein epitopes (Yuen, Wong, Woo & Au2007). Using immunohistochemical analysis, PEPT1protein is found to be constitutively expressed alongthe brush-border membrane of the intestinal mucosain the proximal intestine of the juvenile orange-spotted grouper. The expression of PEPT1 protein isrestricted to absorptive epithelial cells and is moreevident in enterocytes migrating towards the tips ofthemucosal folds, whereas the mucus-secreting gob-let cells are negative for PEPT1. Therefore, like inhigher vertebrates (see e.g. Daniel 2004), the PEPT1

protein strongly associates with di¡erentiated andmature absorptive enterocytes.To summarize, all expression data suggest that in

any ¢sh species tested, PEPT1mRNA is primarily ex-pressed at the intestinal level, in the portion of the in-testinal tract that is directly involved in digestion andabsorption.Where competition for di¡erent functionsoccurs in the gut, PEPT1 always marks the portionthat is devoted to digestion and absorption. Expres-sion is restricted to the brush-border membrane ofmature enterocytes. Expression in other tissues is al-ways lower than in the gut. The functional role ofPEPT1in such other tissues is still unknown in ¢sh.

Conclusions

The transport of di- and tripeptides across the intest-inal epithelial barrier is possible in theory bya combi-nation of transcellular and paracellular routes.Transcellular transport of di- and tripeptides acrossteleost ¢sh intestinal barrier can be accounted for bya combination of a carrier-mediated route and appar-ent di¡usion. At the brush-border membrane of ¢shenterocytes, the carrier-mediated route is repre-sented by PEPT1. Absorbed di- and tripeptides areeither hydrolysed to component amino acids withinthe cell before basolateral exit via amino acidtransport systems or leave the cell intact via a notyet well-characterized basolateral transporter. Inintact tissue, paracellular transport of di- and tripep-tides has never been analysed, but in principle itcannot be excluded.Currently, the only teleost ¢sh PEPT1 for which a

molecular and functional characterization is avail-able is the zebra¢sh orthologue. In this respect, themolecular and functional depiction of other ¢shorthologues is mandatory in order to assess whetheror not zebra¢sh PEPT1 represents a suitable para-digm for all ¢sh PEPT1 proteins. The recent molecu-lar cloning of the Atlantic cod PEPT1 and thefunctional description of at least twomore ¢sh ortho-logues, e.g. the crocodile ice¢sh and the Atlantic sal-mon, will help considerably to answer this question.In this context, another aspect, unique for teleost¢sh, has to be taken into consideration. That is, morethan one PEPT1-type peptide transporter might bepresent in the same ¢sh species, due to whole-gen-ome duplication in the ancestral teleost ¢sh lineage(see e.g. Jaillon, Aury, Brunet, Petit, Stange-Tho-mann, Mauceli, Bouneau, Fischer, Ozouf-Costaz, Ber-not, Nicaud, Ja¡e, Fisher, Lutfalla, Dossat, Segurens,

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Dasilva, Salanoubat, Levy, Boudet, Castellano, An-thouard, Jubin, Castelli, Katinka, Vacherie, Bie¤ mont,Skalli, Cattolico, Poulain, De Berardinis, Cruaud, Du-prat, Brottier, Coutanceau, Gouzy, Parra, Lardier,Chapple, McKernan, McEwan, Bosak, Kellis, Vol¡,Guigo¤ , Zody, Mesirov, Lindblad-Toh, Birren, Nus-baum, Kahn, Robinson-Rechavi, Laudet, Schachter,Que¤ tier, Saurin, Scarpelli,Wincker, Lander,Weissen-bach & Roest Crollius 2004).This questionwas raisedfor the ¢rst time by Gonc� alves et al. (2007) after ana-lysing zebra¢sh and spotted green pu¡er¢sh (Tetrao-don nigroviridis) genomes (http://www.ensembl.org).Two partial amino acid sequences encoding forPEPT1-type transporters have been shown for zebra-¢sh, one (GenBank Acc. No. NP_932330) correspond-ing to the already described zebra¢sh PEPT1 (Verriet al. 2003; see Romano, Kottra, Barca, Tiso, Ma⁄a,Argenton, Daniel, Storelli & Verri 2006, SupportingInformation available at the Physiological Genomicsweb site, for a complete description of the corre-sponding gene), and the other classi¢ed with theEnsembl Acc. No. ENSDARP00000044807, and twofor spotted green pu¡er¢sh, one corresponding toGenBank Acc. No. CAG04978, the other to GenBankAcc. No. CAG05928 (Gonc� alves et al. 2007). Althoughthere is no evidence that the newly identi¢ed PEPT1-type transporters are expressed, they have pro-posed to designate the described PEPT1 sequences(namely NP_932330 and CAG04978) as PEPT1aand the newly found teleost-speci¢c sequences (EN-SDARP00000044807 and CAG05928) as PEPT1b.All the expression data available suggest that in

any ¢sh species tested, PEPT1 is primarily expressedat the intestinal level, in segments of the intestinaltract that are directly involved in absorption of pro-tein hydrolysis products, thus representing an idealmarker for recognition and localization of this func-tion along the gut. From the current data, it appearsthat PEPT1mRNA is present before the onset of exo-genous feeding and that after ¢rst feeding its levelcontinues to increase during larval development(Verri et al. 2003; Amberg et al. 2008). However,whether or not PEPT1 expression is regulated byand/or responds to physiological (hormonal, ner-vous, environmental, dietary, etc.) stimuli is not yetknown. The few data available, limited and not con-clusive, indicate slight PEPT1 mRNA levels changesin zooplankton- vs. rotifer-fed Atlantic cod larvae(Amberg et al.2008) and no changes in adult Orientalweatherloaches fed commercial diets either high inprotein or high in carbohydrates (Gonc� alves et al.2007). However, in Oriental weatherloaches, a ten-

dency towards a reduction in the PEPT1mRNA levelscould be observed in fasted with respect to fed ani-mals (Gonc� alves et al. 2007). On the basis of these lastconsiderations, it is reasonable to assume that PEPT1regulation represents the next crucial step towardsthe complete comprehensionof the physiological roleof this transporter in ¢sh.

Acknowledgments

This investigation was supported by grants from theUniversity of Salento (formerly University of Lecce)(Fondi ex-60%) and in part by grants from the Apu-lian Region (Progetto Strategico, cod. Cip PS_070,and Progetto Esplorativo, cod. Cip PE_062). We aregrateful to Dr Ivar R�nnestad for discussion andhelpful suggestions after reading the manuscript.We would also like to thank the journal anonymousreviewers, whose comments have improved thisreview.

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