RESEARCH ARTICLE Activity of intestinal carbohydrases responds to multiple dietary signals in nestling house sparrows

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1 398 The Journl of Experimentl Biology 6, Pulished y The Compny of Biologists Ltd doi:.4/je.864 RESEARCH ARTICLE Ativity of intestinl rohydrses responds to multiple dietry signls in nestling house sprrows Pwe Brz k, *, Kevin D. Kohl,, Enrique Cviedes-Vidl,,3 nd Willim H. Krsov Deprtment of Forest nd Wildlife Eology, University of Wisonsin, 63 Linden Drive, Mdison, WI 5376, USA, Lortorio de Biologí Professor E. Cviedes Codeli, Fultd de Cienis Humns, nd Deprtmento de Bioquími y Cienis Biológis, Fultd de Quími, Bioquími y Frmi, Universidd Nionl de Sn Luis, 57 Sn Luis, Argentin nd 3 Instituto Multidisiplinrio de Investigiones Biológis de Sn Luis, Consejo Nionl de Investigiones Científis y Ténis, 57 Sn Luis, Argentin *Author for orrespondene t present ddress: Deprtment of Biology, University of Bi ystok, wierkow B, 5-95 Bi ystok, Polnd (rzek@uw.edu.pl) Present ddress: Deprtment of Biology, University of Uth, 57 South 4 Est, Slt Lke City, UT 84, USA SUMMARY The dptive modultion hypothesis predits tht tivity of digestive enzymes should mth the mount of their sustrtes in diet. Interestingly, mny psserine irds do not djust the tivity of intestinl rohydrses to dietry rohydrte ontent. It is diffiult to ssess the generlity of this rule, euse in some studies psserines fed on low-rohydrte nd high-lipid diet showed redued tivity of intestinl rohydrses. However, s rohydrse tivity my e inhiited y high dietry lipid ontent, it is unler whether oserved effets refleted lk of indution y the low rohydrte levels or suppression y the high lipid levels. Here, we isolted the speifi effets of dietry rohydrte nd lipid on rohydrses. We hnd-fed house sprrow nestlings on diets with 5% strh nd 8% lipid (diet HS), no strh nd % lipid (HL), or 5% strh nd % lipid (HSL). Our results show tht tivity of intestinl rohydrses is simultneously indued y dietry rohydrtes nd deresed y dietry lipid, lthough the ltter effet seems stronger. Ativities of mltse nd surse summed over the totl intestine deresed in the order HS>HSL>HL. We oserved omplex intertion etween diet omposition nd intestinl position for mss-speifi tivity of these enzymes, suggesting site-speifi responses to hnges in digest omposition long the intestines used y digestion nd sorption. We re-interpret results of erlier studies nd onlude tht there is no unequivol exmple of dptive modultion of intestinl rohydrses y dietry rohydrte in dult psserine irds, wheres the present experiment onfirms tht nestlings of t lest some speies possess suh pity. Key words: phenotypi flexiility, digestive enzymes, house sprrow, eologil physiology, ontogeny. Reeived 9 Jnury 3; Aepted 5 July 3 INTRODUCTION Morphology nd physiology of the gstrointestinl trt show severl dpttions to different food types, hitts nd life strtegies (reviewed in Krsov nd Hume, 997; Strk nd Wng, 5; Krsov nd Mrtinez del Rio, 7; Krsov et l., ). Presumly, ny unneessry spre digestive pity involves osts of mintining exess orgns or enzymes. In ontrst, insuffiient digestive pity ould llow some energy nd nutrients to espe the gstrointestinl trt unutilized. Thus, nturl seletion is expeted to mth pity of the digestive system to nimls needs nd onditions. This n e espeilly true for intestinl digestive enzymes tht ply key role in food hydrolysis, ut t the sme time must shre limited spe in intestinl epithelium. The dptive modultion hypothesis predits tht the tivity of digestive enzymes should e mthed to reltive levels of their dietry sustrtes (Krsov nd Dimond, 988). The vin digestive system is hrterized y high phenotypi flexiility to ope with high energeti osts nd physil onstrints of flight, rpid growth rte or long migrtion (reviewed in MWhorter et l., 9). Mny ird speies hve vrile diet or show ontogeneti or sesonl swithes in their food hits. Thus, it is resonle to expet tht irds should e le to djust tivity of their intestinl digestive enzymes to diet omposition. However, the pttern tht emerges from previous studies is tht in dult psserine irds, there is n inter-speifi orreltion etween diet omposition nd tivity of intestinl rohydrses, ut not proteses (Kohl et l., ; Rmirez-Otrol et l., ), wheres on n intr-speifi level dult psserines re le to djust tivity of intestinl proteses to diet omposition, ut not rohydrses (reviewed in MWhorter et l., 9). In ontrst, dult glliforms nd nseriforms seem le to modulte tivity of their rohydrses ut usully do not modulte proteses (MWhorter et l., 9). Desried ptterns suggest the presene of fundmentl, phylogeneti differenes in the strtegy for flexiility of intestinl enzyme tivities in irds. However, two studies on psserine irds dult pine wrlers (Dendroi pinus) (Levey et l., 999) nd nestling house sprrows (Psser domestius) (Brz k et l., 9; Brz k et l., ) found signifint, dptive modultion of mltse tivity y dietry rohydrtes. This is prtiulrly puzzling euse neither dult yellow-rumped wrlers (Dendroi oront, ongener of the pine wrler) (Afik et l., 995) nor dult house

2 398 The Journl of Experimentl Biology 6 () HL diet % strh % lipid 6% protein HS diet 5% strh 8% lipid 46% protein Test for indution y strh Test for inhiition y lipid HSL diet 5% strh % lipid 34% protein Fig.. Different experimentl diets nd preditions of the indution y strh nd inhiition y lipid hypotheses. sprrows (Cviedes-Vidl et l., ) show suh flexiility. However, oth mentioned studies mnipulted the ontents of oth rohydrte nd lipids in the diet. In lortory rodents, high levels of dietry lipids derese tivity of rohydrses (MCrthy et l., 98; Tkse nd God, 99; God nd Tkse, 994), nd there is evidene of this in dult house sprrows (Cviedes-Vidl et l., ), ut this effet hs never een refully tested in irds. Beuse of different dietry lipid ontent, the enzyme flexiility oserved in dult pine wrlers nd young house sprrows ould e explined y n inrese in tivity of rohydrses y rohydrtes in irds fed on high rohydrte/low lipid diet ( indution y strh ), y derese in tivity of rohydrses y lipids in irds fed on low rohydrte/high lipid diet ( inhiition y lipid ), or y omintion of oth mehnisms (ompre God nd Tkse, 994; God et l., 995). The gol of the present experiment ws to isolte the speifi effets of dietry rohydrte nd of dietry lipid on rohydrse tivities in nestlings of the house sprrow (Psser domestius L.). Young house sprrows show grdul inrese in the onsumption of high-strh dietry items (e.g. seeds) during ontogeny (Anderson, 6), whih is ompnied y simultneous inrese in the tivity of pnreti nd intestinl enzymes involved in the digestion of strh (Cviedes-Vidl nd Krsov, ; Brz k et l., 9; Brz k et l., ; Brz k et l., 3). We hnd-fed young house sprrows under lortory onditions on three diets: highstrh nd low-lipid (herefter referred to s HS), strh-free nd high-lipid (HL), nd diet tht omined the strh ontent of diet HS with the lipid ontent of diet HL (herefter HSL). By ompring enzyme tivity in house sprrow nestlings rised on these diets, we ould distinguish etween oth hypotheses explining the effet of high rohydrte low lipid diet on rohydrses (Fig. ). If tivity of rohydrses is indued y higher strh ontent, then we should oserve tht irds fed on diet HSL should hve higher tivity of these enzymes thn irds fed on diet HL with the sme lipid ontent ut without strh. However, if the tivity of rohydrses is redued y high lipid ontent, we predit tht irds fed on diet HSL should hve lower tivity of these enzymes thn irds fed on diet HS with the sme strh ontent ut lower lipid ontent. MATERIALS AND METHODS Animl mintenne nd experimentl tretments Two experimentl groups used in the present experiment were prt of the study desried in our previous pper (Brz k et l., ), nd methods used in the present experiment re identil s those desried there, whih were in turn sed on methods used in Brz k et l. (Brz k et l., 9). Briefly, 3-dy-old house sprrow nestlings (dy =dy of hth) were olleted in My July 8 from nturl nests loted on the mpus of the University of Wisonsin, Mdison, nd housed individully in our lortory. Nestlings were hnd-fed using three syntheti diets. One group of nestlings ws fed strhfree nd high-lipid diet (HL), intended to mimi insets onsumed y very young house sprrows, nd nother group ws fed strhontining diet (HS), intended to mimi the mixture of insets nd seeds typil of older nestlings (see Tle for their ompositions). These two groups re the sme s nestlings disseted in dy in our previous study (Brz k et l., ). HL nd HS diets were referred to, respetively, s nd + in our previous ppers (Brz k et l., 9; Brz k et l., ), ut in the present study we ssigned them new revitions tht etter explin differenes etween diets. In the present experiment, we dded third group of nestlings tht were rised together with the two others groups ut were fed diet tht omined the strh ontent of the HS diet nd lipid ontent of the HL diet (herefter referred to s HSL; Tle ). To ontrol for nest effet, when more thn one nestling ws olleted from the sme nturl nest, individuls were rndomly ssigned to different experimentl diets. The smple size ws 9 in ll three groups. In omprison to previously desried feeding tehniques (Brz k et l., 9), hourly mel sizes offered to nestlings on dys 3 5 were inresed to.4,.55 nd.65 g, respetively. Food intke did not differ etween the three tretment groups over the entire experimentl period (ANOVA, P=.3). When nestlings rehed dy, they were killed with CO, nd disseted to remove nd weigh the intestines, gizzrd, liver, pnres nd petorl musles. Intestines were flushed with ie-old vin Tle. Composition of diets used in the present study HL (% of dry mss) HS (% of dry mss) HSL (% of dry mss) Corn strh Csein (protein) Corn oil 8 Alphel non-nutritive ulk Sili snd Amino ids, vitmins, minerl slt, et.* HL, high-lipid nd strh-free diet; HS, low-lipid nd high-strh diet; HSL, high-lipid nd high-strh diet. Diets were mixed with distilled wter t rtio y mss of :3 (diet:wter). *Content s desried y Lepzyk et l. (Lepzyk et l., 998).

3 Diet omposition nd digestive enzymes 3983 Ringer s solution to ler them nd ut into three setions, orresponding to the proximl, medil nd distl regions of intestine, whih were weighed gin (eh setion seprtely) nd immeditely preserved in liquid nitrogen. All experimentl proedures were pproved y the University of Wisonsin, Mdison, ethis ommittee (permit no. RARC A ). Intestinl enzyme ssys To test our preditions we quntified the rte of hydrolysis of mltose, surose nd L-lnine-p-nitronilide. Mltose hydrolysis (i.e. mltse tivity) quntifies the omined tivity of three intestinl rushorder memrne (BBM) enzymes ginst sustrtes of strh hydrolysis. Two of these enzymes mltse, whih hydrolyzes the dishride mltose, nd gluomylse, whih hydrolyzes di-, trind tetr-shrides (ll with -,4 gluose-gluose onds) re oded for y the sme gene omplex MGAM (Quezd-Clvillo et l., 7). The third enzyme, isomltse, whih hydrolyzes -,4 nd -,6 gluose-gluose onds, is oded for y the gene omplex SI (surse-isomltse) (Quezd-Clvillo et l., 7). SI lso odes for surse tivity ginst the -, gluose-frutose ond of surose. Hydrolysis of L-lnine-p-nitronilide quntifies the tivity of the BBM enzyme minopeptidse-n. We used homogentes of intestinl tissue, whih yield mesure with high repetility of the tivity of BBM enzymes. In mie, there is no signifint differene etween surse tivity mesured in tissue homogentes nd in everted sleeves tht isolte the mesurement to the BBM (Lee et l., 998). Thus, the homogente likely gives good indition of BBM tivity, nd there does not seem to e n overestimte due to lot of other possile intrellulr soures of tivity. Also, nother method involving isoltion of BBM vesiles my result in n underestimtion of enzymti pity euse of ineffiient reovery during the isoltion proess (Mrtinez del Rio, 99). Assys of intestinl digestive enzymes were performed s desried in detil elsewhere (Brz k et l., 9), with two modifitions: () intestinl smples nlyzed in previous study (Brz k et l., 9) represented only prt of eh intestinl setion (proximl, medil nd distl), ut in the present experiment we ut intestines from eh setion longitudinlly nd used one hlf of eh setion for enzyme ssys (the other ws preserved for other ssys), resulting in etter nlysis of enzyme tivity over the whole intestine length; nd () in ddition to mltse nd minopeptidse- N, we lso nlyzed surse tivity. The surse ssy ws identil to the mltse ssy desried in Brz k et l. (Brz k et l., 9), exept surose ws sustituted s the sustrte. We expressed mss-speifi tivity of enzymes in eh intestinl setion s miromoles of sustrte proessed per minute per grm of wet tissue. We then lulted the summed hydrolysis tivity of the entire smll intestine, whih is n index of the totl hydrolyti pity, y multiplying mss-speifi tivity in the proximl, medil nd distl intestinl regions y their respetive msses, nd summed over the three regions. Finlly, we lulted the rtio of vlues found for mltse to tht of minopeptidse-n (herefter M/A rtio) for oth mss-speifi nd summed enzymti tivities. This rtio represents reltive investment in rohydrte- nd proteindigesting enzymes nd is not ffeted y potentil differenes in intestinl morphology or villus re etween groups. Dt nlysis Dt were nlyzed y mens of one-wy ANOVA/ANCOVA, with diet tretment s the min ftor. For orgn size, initil nd/or finl ody mss were inluded in the model s ovrites when they were signifint. Repeted-mesures ANOVA ws used to exmine the effet of diet nd intestinl position (proximl, medil, distl) on mss-speifi enzyme tivities. We inspeted the distriution of tested vriles nd rried out Brtlett s nd Levene s tests to hek for homogeneity of vrine. Mss-speifi nd summed tivities of mltse, s well s vlues of the M/A rtio, were log-trnsformed to improve homogeneity of vrine (however, non-trnsformed dt re depited in figures). All tests were rried out using SAS softwre (SAS Institute, Cry, NC, USA). In ll tests, the signifine level ws set t P<.5. We did not djust our signifine level for multiple omprisons, ut relied mostly on the M/A rtio for interprettion of our results, whih offers single estimte of reltive investment into rohydrte nd protein digestion intestinl enzymes. RESULTS Experimentl groups did not differ in their initil ody mss on dy 3, the finl ody mss on dy or the size of ny prt of the digestive system (liver, pnres, gizzrd, intestines) or petorl musle mesured on dy (for ll vriles, effet of diet, P>.5). Summed mss-speifi enzyme tivities Diet tretment exerted signifint effet on the summed tivities of mltse nd surse, s well s on the M/A rtio of summed enzyme tivities (Tle, Fig. ). For ll these vriles, the diet effet followed the sme pttern: nestlings fed on the HS diet hd higher vlues of studied prmeters thn those fed on the HL nd HSL diets [lest signifint differene (LSD) test; for M/A rtio in HS versus HSL irds, P=.3, for ll other omprisons, P<.5], nd nestlings fed on the HSL diet hd higher vlues thn those fed on the HL diet (LSD test, P<. for ll omprisons). In ontrst, summed tivity of minopeptidse-n ws not signifintly ffeted y dietry tretment (Tle, Fig. C). Mss-speifi enzyme tivity in different intestine setions Repeted-mesures ANOVA reveled the presene of highly signifint intertion etween diet tretment nd intestinl position for mss-speifi tivity of mltse, surse nd M/A rtio (Tle 3). Therefore, we nlyzed the effet of diet seprtely for eh setion of intestine y mens of one-wy ANOVA (Tle ), followed y LSD test to identify sttistil differenes etween diets. Nestlings fed on the HS diet hd signifintly higher vlues of mss-speifi tivities of mltse, surse nd the M/A rtio in the proximl nd medil prt of the intestines thn nestlings fed on the two other diets (P<.5 for ll omprisons; Tle, Fig. 3). There ws no signifint differene etween the HSL nd HL diets for these prmeters in the proximl prt of the intestines (P>.5 for ll omprisons). In the medil prt of the intestines, nestlings fed on the HSL diet hd higher mss-speifi tivity of mltse (P=.7) ut not surse (P=.), s well s higher vlues of the M/A rtio (P=.6) thn those fed on the HL diet. Finlly, the effet of dietry mnipultion in distl setion of intestines ws very different from tht oserved in the proximl nd medil setions, euse nestlings fed on the HSL diet showed higher mss-speifi tivity of oth dishridses nd vlues of the M/A rtio thn nestlings fed on the HS or HL diets (P<. for ll omprisons; Tle, Fig. 3). Nestlings fed on the HS nd HL diets did not differ in mss-speifi tivities of mltse nd surse in the distl setion of the intestines (P>.5 for oth omprisons), ut the HS-fed group hd higher M/A rtios (P=.43).

4 3984 The Journl of Experimentl Biology 6 () Tle. Summry of results of ANOVA for the effet of dietry tretment on summed enzyme tivity nd mss-speifi enzyme tivity in different intestinl setions Mltse Surse minopeptidse-n M/A rtio F,4 P F,4 P F,4 P F,4 P Summed tivity < < <. Proximl Medil 3.86 <. 4.6 < <. Distl 7.64 < < <. M/A rtio, mltse/minopeptidse-n rtio. The intertion etween diet tretment nd intestinl position ws not signifint for mss-speifi tivity of minopeptidse-n (Tle 3). Nevertheless, we rried out seprte one-wy ANOVAs independently for eh setion of the intestines for omprison to trends oserved in rohydrses. Diet hd signifint effet only in medil setions (Tle, Fig. 3C): nestlings fed on the HSL diet hd lower mss-speifi tivity of minopeptidse-n thn HL nd HS nestlings (P<. for oth omprisons), wheres the ltter two groups did not differ (P=.5). DISCUSSION Ptterns of the effet of diet omponents on tivities of intestinl enzymes Diet omposition signifintly modulted the tivity of intestinl rohydrses in studied house sprrow nestlings. Summed tivities of mltse nd surse, s well s vlues of the M/A rtio lulted for summed tivities, hnged in the order HS>HSL>HL (Tle, Fig. ). These results onfirm preditions of oth the indution y strh nd inhiition y lipid hypotheses (see Fig. ). However, highly signifint intertion etween dietry tretment nd intestinl position for mss-speifi tivities of dishridses nd the M/A rtio suggests tht the effet of different diet omponents on studied prmeters hnged in proximl-to-distl grdient (Tle 3, Fig. 3). In the proximl setion of the intestines, nestlings fed on the HS diet hd signifintly higher mss-speifi tivities of mltse nd surse, nd higher vlues of the M/A rtio thn irds fed on the lipid-rih HL nd HSL diets, wheres nestlings fed on the HL nd HSL diets did not differ (Fig. 3). This pttern onfirms the predition of only the inhiition y lipid hypothesis (see Fig. ). In the medil setion of the intestines, mss-speifi tivity of mltse nd vlues of the M/A rtio hnged in the order HS>HSL>HL (Fig. 3), pttern tht onfirms preditions of oth tested hypotheses. However, mssspeifi tivity of surse ws not elevted signifintly y strh in this setion (Fig. 3B), nd the prohiitive effet of lipid seems stronger thn the effet of strh (Fig. 3A; hnges in vlues of the M/A rtio in the group fed on the HSL diet prtly refleted hnges in tivity of minopeptidse-n, f. Fig. 3C,D). Finlly, in the distl prt of the intestines, the mss-speifi tivities of rohydrses nd vlues of the M/A rtio were higher in nestlings fed on the HSL diet thn in those fed on the HS nd HL diets (Fig. 3). As we desrie elow, this unexpeted pttern n lso e explined y oth hypotheses. The effet of diet omposition on minopeptidse-n tivity ws muh weker thn in the se of rohydrses, n expeted result euse reltive hnges in protein ontent of experimentl diets were lower thn for strh ontent (Tle ). Nevertheless, the deline in mss-speifi tivity of minopeptidse-n in the order Summed mltse tivity (μmol min ) A Summed surse tivity (μmol min ).5.5 B Fig.. Summed tivity of mltse (A), surse (B), minopeptidse-n (C) nd the rtio of summed tivity of mltse to tht of minopeptidse-n (D) in the present experiment. Mens ± s.e.m. re shown. Different letters indite signifint differenes etween diets. Summed minopeptidse N tivity (μmol min ) C M/A rtio D

5 Diet omposition nd digestive enzymes 3985 Tle 3. Summry of results of repeted-mesures ANOVA for intestinl enzyme mss-speifi tivities nd mltse/minopeptidse-n rtio (M/A rtio) Diet Intestinl position Diet intestinl position F,4 P F,48 P F 4,48 P Mltse 4.68 < <..5 <. Surse 8.46 < <..89 <. minopeptidse-n < M/A rtio 3. < < <. HL>HS>HSL (Fig. 3C) ws in greement with the derese in dietry protein ontent of these diets, ut we reiterte tht the differenes in minopeptidse-n tivity usully were nonsignifint nd were not lrge enough to ffet summed enzyme tivity (see Fig. C). Thus, tivity of minopeptidse-n ws proly wekly indued y dietry protein ut did not depend on dietry lipid (HL nd HSL diets with the sme lipid ontent tended to show, respetively, the highest nd lowest tivity of minopeptidse-n). Similrly, omposition of dietry lipids inhiited tivity of rohydrses ut not leuine minopeptidse in piglets (Dudley et l., 994). Moleulr sis for oserved dietry modifition of intestinl enzymes Diet n modulte tivity of digestive enzymes on intestinl epithelium either y () non-speifi effets, suh s those on morphology of villi, mirovilli, fluidity of ell memrnes, et., whih might simultneously ffet severl enzymes in the sme fshion, or () speifi effets, suh s those on the rte of expression or degrdtion of prtiulr enzyme t the BBM. Beuse hnges in mss-speifi tivities of rohydrses nd minopeptidse-n in our experiment differed in oth diretion nd mgnitude (Fig. 3), we hypothesize tht oserved diet effets were proly medited y site-speifi hnges in the rte of synthesis or degrdtion of rohydrses. Studies in rodents reveled severl geneti nd moleulr mehnisms tht n e responsile for suh hnges (Tkse nd God, 99; Shinohr et l., 993; Kishi et l., 999; God, ; Honm et l., 7; Tnk et l., 8; Mohizuki et l., ; Mohizuki et l., ). We nnot pinpoint the mehnisms tht were responsile for dietry modultion of intestinl rohydrse tivities oserved in our experiment. However, our results suggest tht the reltive strength of the opposite effets of dietry strh nd lipid on the expression of dishridses hnges in proximl-to-distl grdient, s shown y signifint intertion etween diet omposition nd intestinl setion (Fig. 3). Dietry mnipultions often result in suh regionlly speifi hnges in enzyme tivities (Krsov nd Hume, 997). We hypothesize tht the intertion etween diet omposition nd intestinl setion reflets hnges in onentrtion of luminl nutrients used y their grdul hydrolysis nd sorption. Digestion of rohydrtes in the proximl nd medil prts of intestines might e less effiient in HSL-fed thn in HS-fed nestlings euse tivities of dishridses were suppressed here y high lipid ontent in the HSL diet. Therefore, onentrtion of rohydrtes in the distl setion of the intestines in HSL-fed nestlings might e higher thn in those fed on the HS diet. At the sme time, s lipid onentrtion in the intestinl lumen presumly deresed grdully (in ll groups) euse of its digestion nd sorption, its inhiitive effet on expression of dishridses might e weker in the distl thn Mss-speifi mltse tivity (μmol g tissue min ) Mss-speifi minopeptidse N tivity (μmol g tissue min ) A HS HSL HL Mss-speifi surse tivity (μmol g tissue min ) Proximl Middle Distl Proximl Middle Distl C Proximl Middle Distl M/A rtio B D Proximl Middle Distl Fig. 3. Mss-speifi tivity of mltse (A), surse (B), minopeptidse-n (C) nd the rtio of mss-speifi tivity of mltse to tht of minopeptidse-n (D) in the present experiment. Mens ± s.e.m. re shown. Squres nd solid lines indite the group fed on the HS diet, tringles nd dotted lines indite the group fed on the HSL diet, nd irles nd dshed lines indite the group fed on the HL diet. Different letters indite signifint differenes etween diets (tested independently within eh intestinl position).

6 3986 The Journl of Experimentl Biology 6 () in the proximl nd medil setions of the intestines. As result of these two ftors, HSL-fed irds showed higher tivity of mltse nd surse in the distl prt of the intestines thn nestlings fed on the HS diet. However, even though HL-fed nestlings might show similr proximl-to-distl dereses in onentrtion of luminl lipids, omplete lk of strh in their diet did not upregulte expression of rohydrses in the distl setion of the intestines. Interestingly, redution in surse tivity used y high ontent of dietry orn oil my lso e wekest in the distl prt of rt intestines, s suggested y fig. in Tkse nd God (Tkse nd God, 99). We nnot exlude the presene of other mehnisms, suh s lower suseptiility of the distl epithelium to the inhiitory effets of lipids. Moreover, we must dd three vets to our interprettion of otined results. First, our tests ssume impliitly tht differenes in protein ontent mong pplied diets (Tle ) did not ffet the tivity of dishridses. However, oserved ptterns in enzyme tivities do not suggest tht dietry protein ffets tivity of dishridses or tht dietry strh/lipid ffet tivity of minopeptidse-n (f. Figs, 3 nd Tle ). Seond, sustrte onentrtion in the intestinl lumen depends not only on diet omposition ut lso on tivity of pnreti enzymes tht rry out the first steps of food hydrolysis. However, we hypothesize tht tivity of pnreti enzymes my not e limiting. The humn pnres my possess n enormous, tenfold exess pity (DiMgno et l., 973), whih represents one of the highest sfety ftors oserved in ny iologil struture [see fig. 6 in Piersm nd vn Gils (Piersm nd vn Gils, )]. Whether the sme holds in irds is not known, ut one feture of regultion of their digestive enzymes is onsistent with this pttern. We found little evidene for the presene of dptive, diet-relted modultion of pnreti mylse in oth psserine nd gllonserine speies (Brz k et l., 3). Tht study inluded the sme house sprrow nestlings tht were the sujet of the present experiment, nd tivity of their pnreti mylse did not differ signifintly ross the HS nd HL diets. Third, our mltse ssy quntified the tivity of severl enzymes (see Mterils nd methods), nd their reltive roles in digestion of rohydrtes is still not fully understood even in mmmls (see Quezd-Clvillo et l., 7). However, we think tht our mltse ssy offers relile estimte of the funtionl tivity of ll the BBM enzymes tht proess produts of strh hydrolysis y pnreti mylse. Moreover, euse diet omposition modulted oth mltse tivity nd surse tivity in the sme wy, it lso seems likely tht oth genes tht re responsile for mltse tivity (MGAM nd SI) were ffeted y diet similrly. Finlly, we do not know how different ptterns of expression of intestinl dishridses ffet digestion of strh. House sprrow nestlings fed on the HS diet tended to hve etter effiieny of strh ssimiltion thn nestlings fed on the HL diet, ut this effet ws virtully sent in older nestlings [see fig. 6A in Brz k et l. (Brz k et l., 9)]. Moreover, diet omposition usully hd no detetle effet on growth nd development of nestlings (Brz k et l., 9; Brz k et l., ; present study). Thus, different summed tivity nd different proximl-to-distl grdients of mss-speifi tivity of intestinl rohydrses hve no effet on development of young sprrows, t lest under lortory onditions. Implitions for interprettion of other studies In some studies tht investigted the effet of diet omposition on tivity of intestinl enzymes in psserine irds, lipid ontent of experimentl diets ws kept onstnt (Mrtinez del Rio et l., 995; St et l., 998). Two studies on Dendroi wrlers, however, used nturl diets tht were either rih in rohydrtes ut with low lipid ontent (fruits), or hd reltively low rohydrte nd high lipid ontent (insets nd seeds). Diet did not ffet mssspeifi tivity of rohydrses in yellow-rumped wrlers (Afik et l., 995), ut pine wrlers fed on fruit diet hd higher tivity of mltse nd surse thn irds fed on insets nd seeds (Levey et l., 999). However, results of the present experiment show tht identifying the dietry signl for the ltter result is equivol s it mthes preditions of oth indution y strh nd inhiition y lipids hypotheses. Finlly, dult house sprrows fed on high lipid nd low strh diet hd lower tivity of mltse nd surse thn irds fed on diets with low lipid nd either low or high strh ontent (Cviedes-Vidl et l., ), pttern tht supports the inhiition y lipid ut not the indution y strh hypothesis. None of the ited studies found signifint intertions etween diet omposition nd intestinl position for tivity of rohydrses. However, we re not wre of ny previous experiment tht inluded diet similr to the HSL diet in our study, i.e. omining high lipid with high rohydrte ontent nd, s we explined erlier, suh omintion is prtiulrly likely to produe omplex intertions etween intestinl position nd mss-speifi tivity of the studied enzymes. In onlusion, this is, to the est of our knowledge, the first study tht hs shown unequivolly tht dietry rohydrte indues tivity of intestinl dishridses in ltriil irds, lthough this effet is most pronouned when the level of dietry lipid is low (i.e. the effet of lipid is stronger thn tht of rohydrtes). However, there re some importnt issues tht must e solved efore we n generlize our findings. First, the HSL nd HL diets in the present experiment were rih in highly unsturted orn oil, whih is not typil of ll nturl diets. In mmmls, the effet of lipid on intestinl rohydrses my depend on lipid type (Tkse nd God, 99; Dudley et l., 994; Ysutke et l., 995). Seond, speies my differ in their suseptiility to the effet of lipid [t lest in mmmls; see Dudley et l. (Dudley et l., 994) nd disussion there]. Finlly, our study my explin the single outlier mong studies on the effet of diet on intestinl rohydrses in dult psserines (i.e. Levey et l., 999), nd thus support the hypothesis out the lk of dptive modultion of these enzymes in dult psserine irds. However, the present experiment simultneously onfirms tht dietry strh indues intestinl rohydrses in house sprrow nestlings. Thus, it is likely tht t lest some young psserine irds n possess developmentl flexiility of intestinl rohydrses tht is lost lter in life (Brz k et l., ; f. Toloz nd Dimond, 99). BBM HL HS HSL LIST OF ABBREVIATIONS rush-order memrne high-lipid nd strh-free diet low-lipid nd high-strh diet high-lipid nd high-strh diet ACKNOWLEDGEMENTS We re very grteful for Brett Bsler, Heidi Bissell, Twny Cry, Rhel Colpert, Mihel Connolly, Cndie Hskin, Keeshi Hoefer, Lilin Person nd Shin Stewrt for their skillful help in lorious hnd-feeding of our nestlings. We thnk the stff of the Diry Cttle Center, University of Wisonsin, Mdison, for permitting ess to nests in their filities. AUTHOR CONTRIBUTIONS P.B., E.C.-V. nd W.H.K. designed the reserh; P.B. onduted the reserh, rried out sttistil nlyses of the dt nd wrote the first drft of the rtile; K.D.K. rried out iohemil ssys; ll uthors revised nd pproved the rtile.

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