Title. Author(s) Hara, Hiroshi; Akatsuka, Naoki; Aoyama, Yorita. Citation The Journal of Nutritional Biochemistry, 12(8) Issue Date

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1 Title Non-essentil mino cids ply n importnt ro nitrogen feeding. Author(s) Hr, Hiroshi; Aktsuk, Noki; Aoym, Yorit Cittion The Journl of Nutritionl Biochemistry, 12(8) Issue Dte Doc URLhttp://hdl.hndle.net/2115/15872 Type rticle (uthor version) File Informtion JNB12-8.pdf Instructions for use Hokkido University Collection of Scholrly nd

2 Non-essentil mino cids ply n importnt role in dpttion of the rt exocrine pncres to high nitrogen feeding Hiroshi Hr, Noki Aktsuk nd Yoritk Aoym Division of Applied Bioscience, Grdute School of Agriculture, Hokkido University Spporo , Jpn Running title Adpttion of the exocrine pncres Key words Dietry mino cids, non-essentil mino cids, exocrine pncres, trypsin, mylse, rts Corresponding uthor: Hiroshi Hr Division of Applied Bioscience, Grdute School of Agriculture, Hokkido University Kit-9, Nishi-9, Kit-ku, Spporo , Jpn Phone: FAX: or E-mil: hr@chem.gr.hokudi.c.jp 1

3 Abstrct We hve previously demonstrted tht feeding diet with high mino cid (60% AA diet) content, s mixture simulting csein, induced pncretic growth nd pncretic protese production in rts. In the present study, we exmined the effects of n incresing dietry content of essentil mino cids (EAA, x1- x3 in exp. 1 nd x1 - x3.3 in exp. 2) nd non-essentil mino cids (NEAA, x1 - x3 in exp. 1 nd x1 - x5.2 in exp. 2) on pncretic growth, mylse nd protese dpttion using csein-type mino cid mixtures (exp.1, bsl diet; 20% AA diet) nd egg white-type mino cid mixtures (exp.2, bsl diet; 12% AA diet). Pncretic growth nd trypsin ctivity were induced s the dietry content of NEAA ws incresed in experiments 1 nd 2. Amylse ctivity in the pncres ws lso induced s the dietry content of NEAA ws incresed, even with the decrese in dietry crbohydrte in experiment 2. The vlues of ll pncretic vribles decresed with the increse in dietry EAA (x2 nd x3) without n increse in NEAA. The chnges in the pncres were coincident with increses in plsm rginine nd lysine concentrtions nd decrese in the plsm lnine concentrtion. In rts fed 60% AA diet (EAA nd NEAA x3), in the cse of which the EAA content ws blnced with the NEAA content, pncretic growth nd protese production incresed nd reched mximum levels s the plsm mino cid concentrtions decresed, except for lnine. These results show tht NEAA, not EAA, re ssocited with induction of pncretic growth nd protese production upon feeding diet with high AA content, nd tht some metbolites my be involved in the induction process. The suppression of pncretic growth nd protese production in rts fed the high EAA diets without blnced NEAA my be ssocited with impirment of mino cid metbolism rther thn the increments in the concentrtion of one or more essentil mino cids. Our results lso suggest tht there is n unknown mechnism or unknown fctors involved in regulting pncretic mylse. 2

4 Feeding high-protein diet induces pncretic growth nd production of pncretic proteses (1,2). Dietry protein in the lumen of the smll intestine competes with endogenous luminl CCK-relesing peptide fctors for degrdtion by pncretic proteses, resulting in the prolonged survivl of the CCK relesing peptides, nd this induces pncretic chnges in rts (3-5). We previously demonstrted tht feeding diet contining n mino cid mixture simulting csein lso induced pncretic growth nd protese production in rts (6,7). The induction is not medited by cholecystokinin secretion (6). In humns nd dogs, two types of mino cids, phenyllnine nd tryptophn stimulte exocrine pncretic secretion through their ction on the intestinl mucos (8,9). However, we hve shown tht feeding diet with high mino cid content does not induce pncretic protese production in the lumen of the smll intestine using chronic pncretico-biliry diverted rts (10). Dietry mino cids my stimulte the exocrine pncres through n unknown mechnism. The ims of the present study were to determine which kinds of mino cids ply role in the dptive responses of the exocrine pncres upon feeding diet with high mino cid content. First, we exmined the effects of csein-type mino cid mixtures with different rtios of essentil nd non-essentil mino cids (EAA nd NEAA) on pncretic growth, mylse nd protese dpttion, becuse feeding diet with certin kinds of individul mino cids in excess is known to be toxic in rts (11,12). We used the sme mino cid mixture simulting csein s tht used in ll previous work described bove. In experiment 2, we exmined the effects of feeding diet with high mino cid content on the exocrine pncres using n mino cid mixture simulting egg white s nother type of mixture. Experimentl Methods Animls nd diets Mle Wistr-ST rts (Jpn SLC Inc., Hmmtsu, Jpn), weighing bout 100 g, were fed diets contining mixture of mino cids (AA) t norml level, s shown in Fig. 1 (20% AA diet in exp. 1 nd 12% AA diet in exp. 2) (13,14), during the cclimtion period. An mino cid mixture simulting csein ws used for experiment 1 nd mixture simulting egg white ws used for experiment 2, s shown in Tble 1. In experiment 1, the rts were fed diet contining AA simulting csein with 3

5 norml nitrogen level (20% AA diet) for 7 dys nd were divided into 6 groups of 6 rts on the bsis of body weight. The rts in the 6 groups were given 6 test diets contining vrious AA for 5 dys becuse we previously observed tht pncretic protese ctivity reched mximum level fter 5 dys of feeding high-aa diet. The test diets contined AA with vrious rtios of essentil nd non-essentil mino cids s shown in Fig. 1A. All rts were killed without fst t 10:00-11:00 under pentobrbitl nesthesi (50 mg/kg body weight; Abbott Lbortories, North Chicgo, IL) fter smpling of blood from the ort. The whole pncres ws removed immeditely, frozen in liquid nitrogen, nd stored t - 40 C until subsequent nlyses. We confirmed tht the stomch ws sufficiently filled with chyme in the cse of ech rt. In experiment 2, the rts were fed diet contining AA simulting egg white with low nitrogen level (12% AA diet) for 7 dys nd were divided into 5 groups of 8 rts. In this experiment, we set the AA content t 12% s the lowest, level supporting mximum growth in rts, nd 40% s the highest, level not suppressing food intke or growth of the rts. The rts in the 5 groups were given the 5 test diets contining vrious AA shown in Fig. 1B nd Tble 1. We rrnged test diets in group B nd C s totl mino cid content were pproximtely double nd triple compred with tht in group A with fixed NEAA content, nd rrnged test diets in group C, D nd E s EAA content were double nd triple with fixed totl mino cid content in diet (40%). The rts were killed 7 dys fter the strt of feeding the test diets, s in experiment 1, without smpling of blood from the ort. Other detils were the sme s in experiment 1. This study ws pproved by the Hokkido University Animl Committee, nd the nimls were mintined in ccordnce with the guidelines for the cre nd use of lbortory nimls of Hokkido University. Anlyses Trypsinogen in freeze-dried pncres smples ws ctivted by tretment with enterokinse (Sigm Chemicl Co., St. Louis, MO) t 30 C for 20 min in 15 mmole/l Tris buffer (ph 8.1). Pncretic trypsin ctivity ws mesured spectrophotometriclly using synthetic substrte, Nα-p-toluenesulfonyl-L-rginine methyl ester (TAME) (15). Amylse ctivity in the pncres ws mesured with Procion yellow strch (16). Protein ws mesured by modified version of Lowry's method (17, 18). 4

6 Amino cids in plsm were nlyzed by HPLC s phenyl thiocrbmyl (PTC) derivtives with phenyl isothiocynte (19) fter deproteiniztion with n equl volume of cetonitrile. The HPLC system ws equipped with mini-solvent delivery system, M-600 (Wters), nd Wkopk WS-PTC column (4.0 x 200 mm; Wko Pure Chemicl Industries, Osk, Jpn) Clcultions Trypsin ctivity ws expressed s TAME U/100 g body weight (totl ctivity) in the pncres. One unit of trypsin ctivity ws defined s the mount of ctivity resulting in hydrolysis of 1 µmole of substrte per minute t 30 C. Procion yellow strch, the substrte for the mylse ssy, ws clibrted using purified α-mylse from porcine pncres (Type 1A, Sigm Chemicl Co.) t 37 C. The dt were nlyzed by one-wy nlysis of vrince. The existence of significnt difference between groups ws determined by Duncn's multiple rnge test (20) (P < 0.05, SAS version 6.07, SAS Institute Inc., Cry, NC). Results Body weight gin nd food intke seemed to decrese s result of feeding high-nitrogen or high-eaa diet (Tble 2). In experiment 1, the vlues of both prmeters were lower in the cse of rts fed either of the two test diets contining the highest level of essentil AA (diets E nd F) thn those in the cse of rts fed the diet contining 20% AA. In experiment 2, food intke ws somewht lower in the three high-nitrogen groups (diets C, D nd E) thn in the 12% AA group, however, compring groups fed diets with the sme high AA content, feeding diets contining higher EAA rtio did not ffect food intke. There ws no difference in body weight gin mong the groups in experiment 2. Pncretic dry weight fter feeding the diet with the highest NEAA rtio (diet C) ws higher thn tht fter feeding the diet contining 20% AA (diet A, Tble 3), nd similr tendency ws evident in the cse of the pncretic protein content in experiment 1. The protein content of the pncres ws lower in the high EAA rtio groups (diets D nd E) thn in the other groups. The mylse content ws lower in the groups fed the diets contining high rtio of EAA (diets D nd E) thn in the other groups (Fig. 2A). The trypsin (trypsinogen) content of 5

7 the pncres ws the highest in the 60% AA group. Increses in the NEAA content of the diet, but not the EAA content, resulted in n increse in pncretic trypsin ctivity levels (Fig. 2B). The pncretic trypsin ctivities in the cse of both groups fed the high EAA rtio diets were y levels lower thn those in the other groups. The chnges in plsm mino cid concentrtions tht occurred s result of feeding the test diets re shown in Fig. 3. The plsm rginine concentrtion decresed nd the lnine concentrtion incresed grdully with the incresing NEAA content of the diet (diets B nd C vs diet A). The rginine concentrtion ws very high nd the lnine concentrtion ws low in both of the high EAA rtio groups (diets D nd E). The methionine nd leucine concentrtions were higher s the EAA rtio of the diet incresed (diets D nd E vs diet A). The concentrtion of lnine incresed nd those of other mino cids shown in Fig. 3 decresed s result of ddition of NEAA to the high EAA diet (diet E vs diet F). The serine concentrtion vried in mnner similr to the lnine concentrtion. The lysine concentrtion vried in mnner similr to the rginine concentrtion, except for significntly lower vlue in the cse of the 60% AA group s compred to the 20% AA group. Vline nd isoleucine concentrtions vried in mnner similr to the leucine concentrtion s result feeding the test diet contining 20% AA. Other mino cid concentrtions did not chnge mrkedly (dt not shown). In experiment 2 using AA simulting egg white, incresing the NEAA content resulted in n increse in the vlues of ll pncretic vribles, pncretic dry weight nd protein, levels of mylse nd trypsin ctivity in the pncres (diets A, B nd C in Tble 3 nd Fig. 4). No mrked chnges in the vlues of these vribles were observed upon incresing the rtio of EAA, compring the results for diets with the sme AA content (diets C, D nd E). Discussion We hve previously demonstrted tht n increse in dietry intke of n mino cid mixture simulting csein induced pncretic protese production (7). In the present study, we exmined the role of essentil nd non-essentil mino cids in the diet on dpttion of the exocrine pncres using mino cid mixtures composed of essentil mino cids (EAA) nd non-essentil mino cids (NEAA). Pncretic growth nd trypsin were induced with increses production in the mounts of NEAA in the diets, in both experiments 1 nd 2. In contrst, increses in 6

8 EAA resulted in decrese in the vlues of ll pncretic vribles exmined. These results suggest tht NEAA, not EAA, ply role in induction of the chnges in the exocrine pncres observed upon feeding high-aa diet. In rts fed diet contining high EAA with low NEAA (group D nd E in exp. 1), food intke nd body weight gin were lower thn those on other groups. These lower growth possibly suppressed pncretic vribles shown in the high EAA groups of the present study. However, it is not true in the high EAA nd high NEAA group (60% AA in exp. 1). Also, we presented the results of pncretic vribles s vlues per unit body weight. Moreover, we killed rts in morning nd confirmed the stomchs of ll rts were filled with sufficient chyme. This elimintes the effects of different feeding ptterns mong groups on the pncres, if it were. Some NEAA re known to ffect digestive functions. Glutmine nd glutmic cid re importnt for mintining the intestinl mucos (21,22). Chnges in intestinl function my ffect the exocrine pncres. However, pncretic protese production ws similrly induced in experiments 1 nd 2 in spite of the fct tht there ws no glutmte in the AA mixture used in experiment 2 (Tble 1). Also, the plsm glutmine concentrtion ws not ffected s result of feeding the test diets (dt not shown). These mino cids my not be involved in the induction of protese production. Arginine is potent insulinotropic mino cid (23), nd insulin is importnt to mintin the exocrine pncres (24). However, the plsm rginine concentrtion chnged in reciprocl mnner, compred to the chnges in pncretic mylse nd trypsin ctivity levels. It seems unlikely tht rginine is involved in dpttion of the pncres upon intke of high-aa diet. It hs been proposed tht mino cid sensors re present in the duodenojejunum nd the port-heptic system (25,26). Possibly, mino cids in the diet regulte the exocrine pncres through such mino cid sensors. It is recognized tht dietry crbohydrtes re the inducers of pncretic mylse production (27,28). A decrese in the crbohydrte content of the diet usully results in decrese in mylse production. In experiment 2, mylse production ws induced by incresed ingestion of NEAA even when the crbohydrte content of the diet ws reduced from 80% to 50% (from diet A to diet C). In experiment 1, the mylse ctivity did not increse, but ws 7

9 mintined t constnt level, with ingestion of incresing mounts of NEAA. The mylse ctivity in the bsl group (20% AA diet) in experiment 1 ws high compred with tht in the bsl group (12% AA diet) in experiment 2. A possible reson for the different results obtined in experiments 1 nd 2 is tht the bsl diet in experiment 1 contined greter mount of NEAA thn tht in experiment 2. Amino cids, especilly NEAA, in the bsl diet my be utilized by rts very efficiently in experiment 2, nd the mount of NEAA required for induction of pncretic mylse my be low. Some surplus NEAA metbolites, for exmple, glucogenic intermedites, re possibly involved in the induction of pncretic mylse production. Our findings suggest tht there is n unknown mechnism or unknown fctors involved in regulting pncretic mylse. Amylse in the pncres is lso influenced by nutritionl sttus (29,30). Food intke nd body weight gin were reduced in the EAAx3 group (diet E in exp.1), but not in the EAAx2 group (diet D) in spite of the fct tht the mylse level nd the vlues of other vribles were lower in both of the groups. It is not likely tht impired nutritionl sttus cused by lowered food intke is involved in the suppression of mylse production nd the function of the exocrine pncres. Ingestion of diets with incresing EAA content suppressed pncretic growth nd the levels of pncretic enzyme ctivity. There ws no evidence of such suppression in the cses where the high EAA ws blnced by ddition of NEAA (group F in exp. 1, groups D nd E in exp. 2). It is reported tht intrvenous high mino cid loding suppressed exocrine pncretic secretion (31,32). The plsm rginine concentrtion shown in Fig. 3 ws mrkedly higher in both high-eaa groups (diets D nd E in exp. 1) nd the lnine concentrtion ws lower in these groups, but these chnges did not show dose dependence. These results suggest tht nitrogen metbolism is impired in rts fed diets contining high EAA content without it being blnced with NEAA. Methionine nd leucine concentrtions were lso higher in the EAAx3 group (diet E), but not in the EAAx2 group (diet D), thn in the three EAAx1 groups (diets A, B, nd C). The suppression of the exocrine pncres did not depend on the dietry content of h x chnges in EAA. This finding suggests tht the suppression is ssocited with impirment of mino cid metbolism rther thn the increments of concentrtion of one or more essentil mino cids. Results of the present study possibly show tht feeding the diet contining high EAA without 8

10 blncing NEAA possibly be toxic for the exocrine pncres. In rts fed the high-aa diet (the 60% AA group, diet F), in the cse of which the EAA content is blnced with the NEAA content, pncretic growth nd pncretic protese incresed nd reched mximum levels s the plsm mino cid concentrtions decresed, except for lnine. This shows tht NEAA improve body mino cid metbolism. In experiments 1 nd 2, the increse in pncretic trypsin ctivity observed is likely to depend on the totl AA content of the diet, except for the EAA-excess diets. The level of protese ctivity in rts fed diet A in experiment 2 (12% AA) ws lower thn tht in rts fed diet A in experiment 1 (20% AA). The level of ctivity ws similr mong the rts fed diets C, D nd E in experiment 2, where the mino cid content ws the sme in spite of the fct tht the composition in terms of individul AA ws substntilly different mong these three diets. These findings suggest tht the totl mount of mino cids is fctor controlling pncretic protese if the metbolism of mino cids is not impired. These findings re consistent with the bove specultion tht some mino cid metbolites induce chnges in exocrine pncretic function. We hve shown in previous studies nd in experiment 1 tht feeding high-aa diet contining n AA mixture simulting csein induces pncretic protese production. In the present study, we showed tht ingestion of diet contining high level of n AA mixture simulting egg white lso induces pncretic protese production. This result suggests tht induction of protese production by high-aa diet is common phenomenon, not specific to the AA mixture simulting csein. In conclusion, pncretic enzyme production nd pncretic growth were found to be induced by ingestion of diets contining high level of n AA mixture simulting either csein or egg white. Increses in the levels of non-essentil mino cids, but not essentil mino cids, in the diet re ssocited with this induction process, nd results of the present study suggest tht mino cid metbolites re involved. 9

11 References 1. Grossmn, M.I., Greengrd, H., nd Ivy, A.C. (1942) The effect of dietry composition on pncretic enzymes. Am. J. Physiol. 138, Temler, R. S., Dormond, C. A., Simon, E., Morel, B., nd Mettrux, C. (1984) Response of rt pncretic proteses to dietry proteins, their hydrolystes nd soyben trypsin inhibitor. J. Nutr. 114, Fushiki, T., Kjiur, H., Fukuok, S.-I., Kido, K., Semb, T., nd Iwi, K. (1989) Evidence for n intrluminl meditor in rt pncretic enzyme secretion. Reconstitution of the pncretic response with dietry protein, trypsin nd the monitor peptide. J. Nutr. 119, Li, L., Louie, D., nd Owyng, C. (1989) A cholecystokinin relesing peptide medites feedbck regultion of pncretic secretion. Am. J. Physiol. 256, G430-G Miysk, K., Gun, D., Liddle, R., nd Green, G.M. (1989) Feedbck regultion by trypsin, evidence for intrluminl CCK-relesing peptide. Am. J. Physiol. 257, G175-G Hr, H., Nrkino, H., nd Kiriym, S. (1995) Induction of pncretic growth nd proteses by feeding high mino cid diet does not depend on cholecystokinin in rts. J. Nutr. 125, Hr, H., Hshimoto, N., Aktsuk, N., nd Ksi, T. (2000) Induction of pncretic trypsin by dietry mino cids in rts: four trypsinogen isozymes nd cholecystokinin messenger RNA. J. Nutr. Biochem. 11, Thimister, P.W., Hopmn, W.P., Sloots, C.E., Rosenbusch, G., Willems, H.L., Trijbels, F.J., nd Jnsen, J.B. (1996) Role of intrduodenl proteses in plsm cholecystokinin nd pncreticobiliry responses to protein nd mino cids. Gstroenterology 110, Meyer, J.H. nd Kelly, G.A. (1976) Cnine pncretic response to intestinlly infused protein nd protein digests. Am. J. Physiol. 231, Hr, H., Ohym, S., nd Hir, T. (2000) Luminl dietry protein, not mino cids induces pncretic protese vi CCK in pncretico-biliry diverted rts. Am. J. Physiol. (in press). 10

12 11. Alm, S. Q., Becker, R. V., Stucki, W. P., Rogers, Q. R., nd Hrper, A. E. (1966) Effect of threonine on the toxicity of excess tyrosine nd ctrct formtion in the rt. J. Nutr. 89, Murmtsu, K., Odgiri, H., Morishit, S., nd Tkeuchi, H. (1971) Effect of excess levels of individul mino cids on growth of rts fed csein diets. J. Nutr. 101, Reeves, P.G. (1989) AIN-76 diet, should we chnge the formultion? J. Nutr. 119, Americn Institute of Nutrition. (1977) Report of the Americn Institute of Nutrition d hoc Committee on Stndrds for Nutritionl Studies. J. Nutr. 107, Rick W. (1976). Trypsin. In Methods of Enzymtic Anlysis, Second English Edition. Vol.2 (Bergmeyer, H.U. ed.), pp , Acdemic Press / Weinheim, Verlg Chemie, New York nd London. 16. Jung, D.H. (1980) Preprtion nd ppliction of procion yellow strch for mylse ssy. Clin. Chim. Act 100, Lowry, O.H., Rosebrough, H.J., Frr, A.L., nd Rndll, R.J. (1951) Protein mesurement with the Folin-phenol regent. J. Biol. Chem. 193, Sugwr, K. (1975) Influence of Triton X-100 on protein determintion by the Lowry procedure. Agric. Biol. Chem. 93, Cohen SA, Bidlingmeyer BA, nd Trvin TL. (1986) PITC derivtives in mino cid nlysis. Nture 320, Duncn, D.B. (1995) Multiple rnge nd multiple F tests. Biometrics 11, Reeds, P. J., Burrin, D. G., Stoll, B., nd Jhoor, F. (2000) Intestinl glutmte metbolism. J. Nutr. 130, 978S-982S. 22. Windmueller, H. G., nd Speth, A. E. (1980) Respirtory fuels nd nitrogen metbolism in vivo in smll intestine of fed rts. Quntittive importnce of glutmine, glutmte, nd sprtte. J.Biol. Chem. 255, Floyd, J.C. Jr., Fjns, S.S., Conn, J.W., Knopf, R.F., nd Rull, J. (1966) Stimultion of insulin secretion by mino cids. J. Clin. Invest. 45, Lee, K.Y., Zhou, L., Ren, X.S., Chng, T.-M., nd Chey, W.Y. (1990) An importnt role of 11

13 endogenous insulin on exocrine pncretic secretions in rts. Am. J. Physiol. 258, G268-G Niijim, A. (2000) Reflex effects of orl, gstrointestinl nd heptoportl glutmte sensors on vgl nerve ctivity. J. Nutr. 130, 971S-973S. 26. Jenningros, R. (1982) Vgl unitry responses to intestinl mino cid infusions in the nesthetized ct: puttive signl for protein induced stiety. Physiol. Behv. 28, Giorgi, D., Bernrd, J. P., Lpointe, R., nd Dgorn, J. C. (1984) Regultion of mylse messenger RNA concentrtion in rt pncres by food content. EMBO J. 3, Wicker, C., Puigserver, A., nd Scheele, G. (1984) Dietry regultion of levels of ctive mrna coding for mylse nd serine protese zymogens in the rt pncres. Eur. J. Biochem. 139, Hr, H., Kiriym, S., nd Ksi, T. (1997) Supplementtion of methionine to low soyben protein diet strikingly increses pncretic mylse ctivity in rts. J. Nutr. Sci. Vitminol. 43, Johnson, A., Hurwitz, R., nd Kretchmer, N. (1977) Adpttion of rt pncretic mylse nd chymotrypsinogen to chnges in diet. J. Nutr. 107, Sitoh, Y., Hond, T., Mtsuno, S., Noto, N., Miyshit, E., nd Sto, T. (1979) Effects of eight mino cids on the exocrine nd endocrine pncretic function. Tohoku J. Exp. Med. 129, Stubbs, R. S., nd Stbile, B. E. (1989) Inhibition of the stimulted cnine exocrine pncres by mino cids nd ft. Arch. Surg. 124,

14 Tble 1 Composition of mino cid (AA) mixtures simulting csein or egg white* Amino cid Csein type Egg white type g / kg mino cid mixture Essentil mino cids L -Histidine-HCl L -Isoleucine L -Leucine L -Lysine-HCl L -Methionine L -Phenyllnine L -Threonine L -Tryptophn L -Vline Non-essentil mino cids L -Alnine L-Arginine-HCl L -Asprtic cid L -Asprgine L -Cystine L -Glutmic cid L -Glutmine Glycine L -Proline L -Serine L -Tyrosine *All mino cids used were crystllized preprtions (Ajinomoto Co., Tokyo, Jpn). In the cse of the mino cid mixture simulting egg white, sprtic cid nd glutmic cid were replced with sprgine nd glutmine to prevent cidifiction of the diet with high rtio of non-essentil mino cids. 13

15 Tble 2 Body weight gin nd food intke of rts fed test diets contining mino cid mixtures simulting csein (exp. 1) or egg white (exp. 2), with vrious rtios of essentil to non-essentil mino cids (EAA to NEAA) exp. 1 exp. 2 Diet group Body weight gin Food intke Body weight gin Food intke (EAA) (NEAA) (g / dy) (g / dy) % in diet A exp.1 (8.8) (11.2) 7.60 ± ± 0.50 exp.2 (5.4) (6.6) 7.70 ± ± 0.16 B exp.1 (8.8) (22.4) 7.19 ± b 14.7 ± 0.57 exp.2 (5.4) (20.5) 8.32± ± 0.18 C exp.1 (8.8) (33.6) 7.29 ± b 14.9 ± 0.30 exp.2 (5.4) (34.6) 7.32 ± ± 0.27 b D exp.1 (17.6) (11.2) 7.42 ± ± 0.29 exp.2 (11.9) (28.1) 8.15 ± ± 0.17 b E exp.1 (26.4) (11.2) 5.98 ± b 11.3 ± 0.34 b exp.2 (18.0) (22.0) 8.27 ± ± 0.21 b F exp.1 (26.4) (33.6) 6.15 ± b 11.9 ± 0.34 b P vlues < More detils on diet groups A, B, C, D, E nd F re those shown in Fig. 1. The vlues shown re men ± SEM for 6 rts (exp. 1) or 8 rts (exp.2). Superscript letters re employed to indicte significnt differences; vlues in ech column not shring the sme letter re significntly different (P <0.05) 14

16 Tble 3 Pncretic dry weight nd protein content of rts fed test diets contining mino cid mixtures simulting csein (exp. 1) or egg white (exp. 2), with vrious rtios of essentil to non-essentil mino cids (EAA to NEAA) exp. 1 exp. 2 Diet group Dry weight Protein content Dry weight Protein content (EAA) (NEAA) (mg / 100 g body weight) (mg / 100 g body weight) % in diet A exp.1 (8.8) (11.2) 111 ± 1.6 bc 87.0 ± 3.41 b exp.2 (5.4) (6.6) 103 ± 4.4 c 68.0 ± 2.77 c B exp.1 (8.8) (22.4) 124 ± 4.4 b 94.3 ± 2.70 b exp.2 (5.4) (20.5) 106± 1.6 bc 71.6 ± 1.79 bc C exp.1 (8.8) (33.6) 127 ± ± 5.85 b exp.2 (5.4) (34.6) 115 ± 4.3 b 77.8 ± 3.42 b D exp.1 (17.6) (11.2) 108 ± 4.6 c 74.1 ± 2.44 c exp.2 (11.9) (28.1) 122 ± ± 3.50 E exp.1 (26.4) (11.2) 102 ± 6.8 c 71.1 ± 4.52 c exp.2 (18.0) (22.0) 121 ± ± 2.80 F exp.1 (26.4) (33.6) 129 ± ± 5.0 P vlues <0.001 <0.001 <0.001 <0.001 More detils on diet groups A, B, C, D, E nd F re those shown in Fig. 1. The vlues shown re men ± SEM for 6 rts (exp. 1) or 8 rts (exp.2). Superscript letters re employed to indicte significnt differences; vlues in ech column not shring the sme letter re significntly different (P <0.05) 15

17 Figure legends Fig. 1 Diet groups in the present study. Amino cid mixtures simulting csein nd egg white were used, s shown in Tble 1. Corn oil (50 g/kg diet) contining retinyl plmitte (7.66 µmole/kg diet) nd ergoclciferol ( µmole/kg diet) ws used. The minerl mixture ws prepred bsed on tht estblished t the AIN-76 Workshop held in 1989 (Reeves 1989). It provided (mg/kg diet): C, 4491; P, 2997; K, 3746; Mg, 375; Fe, 100; I, 0.32; Mn, 10.0; Zn, 34.7; Cu, 6.00; N, 4279; Cl, 6542; Se, 1.05; Mo, 1.00; Cr, 0.50; B, 0.50; V, 0.25; Sn, 2.00; As, 1.00; Si, 20.0; Ni, 1.00; F, 2.72; Co, The vitmin mixture ws prepred in ccordnce with the AIN-76 mixture (AIN 1976) except tht mendione nd L-scorbic cid were dded to mke 5.81 µmole/kg nd 284 µmole/kg diet, respectively. In experiment 1, we dded NHCO3 to neutrlize the cidic mino cid mixtures. Fig. 2 Levels of mylse nd trypsin ctivity in the pncres fter feeding diets contining mixture of mino cids simulting csein, with vrious mounts nd rtios of essentil (EAA) nd non-essentil (NEAA) mino cids. The P vlues estimted by one-wy nlysis of vrince were <0.001 in the cse of both vribles. Men vlues not shring letter re significntly different between diet groups (P <0.05). Fig. 3 Amino cid concentrtion in plsm prepred from ortic blood collected fter feeding diets contining n mino cid mixture simulting csein, with vrious mounts nd rtios of essentil (EAA) nd non-essentil (NEAA). P vlues estimted by one-wy nlysis of vrince were <0.001 in the cse of ll vribles. Men vlues not shring letter re significntly different between diet groups (P <0.05). Fig. 4 Levels of mylse nd trypsin ctivity in the pncres fter feeding diets contining n mino cid mixture simulting egg white, with vrious mounts nd rtios of essentil (EAA) nd non-essentil (NEAA) mino cids. P vlues estimted by one-wy nlysis of vrince were for mylse nd for trypsin. Men vlues not shring letter re significntly different between diet groups (P <0.05). 改ページ Fig. 5 Abundnce of cholecystokinin (CCK) nd secretin mrna in the jejunl mucos fter feeding 60% csein diet or 60% mino cid (AA) mixture diet compred with 20% csein diet or 20% AA mixture diet, for 10 dys. Vlues shown re the men nd stndrd error for six rts. P vlues estimted by two-wy nlysis of vrince were 0.020, nd for nitrogen source (S), level (L) nd S x L in the cse of CCK mrna, 0.170, nd for nitrogen source, level nd S x L in the cse of secretin mrna. Men vlues not shring letter re significntly different between diet groups (P<0.05). 16

18 A 100 Oli & others Sucrose EAA NEAA B 100 Oli & others Sucrose EAA NEAA g / kg diet g / kg diet % in diet A B C D E F EAA (x1) (x1) (x1) (x2) (x3) (x3) NEAA (x1) (x2) (x3) (x1) (x1) (x3) Totl AA % in diet A B C D E EAA (x1) (x1) (x1) (x2.2) (x3.3) NEAA (x1) (x3.1) (x5.2) (x4.3) (x3.3) Totl AA Fig. 1

19 Amylse, U/100 g body weight A B b bc 4000 cd d 3000 b d b % in diet A B C D E F % in diet A B C D E F EAA EAA (x1) (x1) (x1) (x2) (x3) (x3) (x1) (x1) (x1) (x2) (x3) (x3) NEAA NEAA (x1) (x2) (x3) (x1) (x1) (x3) (x1) (x2) (x3) (x1) (x1) (x3) Totl AA Totl AA Trypsin, TAME U/100 g body weight Fig. 2

20 Alnine Arginine Methionine Leucine b 4 0 b b µmol/liter bc d d c c c c 10 5 bc c c b c c c c bc 0 EAA NEAA A B C D E F x1 x1 x1 x2 x3 x3 x1 x2 x3 x1 x1 x3 0 A B C D E F x1 x1 x1 x2 x3 x3 x1 x2 x3 x1 x1 x3 0 A B C D E F x1 x1 x1 x2 x3 x3 x1 x2 x3 x1 x1 x3 0 A B C D E F x1 x1 x1 x2 x3 x3 x1 x2 x3 x1 x1 x3 Fig. 3

21 Amylse, U/100 g body weight A B b b 5000 b % in diet A B C D E EAA (x1) (x1) (x1) (x2.2) (x3.3) NEAA (x1) (x3.1) (x5.2) (x4.3) (x3.3) Totl AA b 500 b % in diet A B C D E EAA (x1) (x1) (x1) (x2.2) (x3.3) NEAA (x1) (x3.1) (x5.2) (x4.3) (x3.3) Totl AA Trypsin, TAME U/100 g body weight Fig.4