Food & Function Accepted Manuscript

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1 Food & Function Accepted Mnuscript This is n Accepted Mnuscript, which hs een through the Royl Society of Chemistry peer review process nd hs een ccepted for puliction. Accepted Mnuscripts re pulished online shortly fter cceptnce, efore technicl editing, formtting nd proof reding. Using this free service, uthors cn mke their results ville to the community, in citle form, efore we pulish the edited rticle. We will replce this Accepted Mnuscript with the edited nd formtted Advnce Article s soon s it is ville. You cn find more informtion out Accepted Mnuscripts in the Informtion for Authors. Plese note tht technicl editing my introduce minor chnges to the text nd/or grphics, which my lter content. The journl s stndrd Terms & Conditions nd the Ethicl guidelines still pply. In no event shll the Royl Society of Chemistry e held responsile for ny errors or omissions in this Accepted Mnuscript or ny consequences rising from the use of ny informtion it contins.

2 Pge 1 of 6 RSCPulishing Cite this: DOI: 1.139/xxxx Received th Jnury 12, Accepted th Jnury 12 DOI: 1.139/xxxx Introduction It is widely ccepted tht the dipeptidyl-peptidse (DPP)-IV inhiitors cn e used s novel tretment for type 2 dietes 1, 2. DPP-IV inhiitors cn protect incretin, especilly glucgonlike peptide-1 (GLP-1) which my decrese lood glucose nd improve impired glucose tolernce, ginst the degrdtion y DPP-IV 3,4. There hve een severl DPP-IV inhiitors, e.g. sitgliptin, vildgliptin, sxgliptin nd logliptin, s the ntidietic gents pproved y United Sttes nd Europe governments. These DPP-IV inhiitors were exmined their ntidietic effects in vivo y niml nd clinicl experiments 5-8. Although the risks of infection nd hypoglycemi for longterm gliptin tretment re low, further reserches re needed to clrify the possile link on other side effects, e.g. stheni, crdic nd vsculr events 9. A DPP-IV inhiitor, 1-[2-[(5-cynopyridin-2-yl) mino] ehtylmino] cetyl-2-cyno-(s)-pyrrolidine monohydrochloride slt (NVP-DPP728) ws used to exmine its effect on the improvement of glycemic control in oese Zucker rts 1. The complete inhiition of plsm DPP-IV ctivity y orlly dministered NVP-DPP728 (1 µmole/kg) t -3 min of the experiment resulted in nd 3% reduction of sl plsm glucose nd insulin concentrtions, respectively, in oese rts. The sme study lso reported tht during n orl glucose tolernce test, tretment of NVP-DPP728 completely inhiited plsm DPP-IV ctivity, highly preserved the endogenously secreted GLP-1, ugmented the insulin response nd ll ut restored the glucose excursion to norml in glucose intolernt, oese Zucker rts. Another study exmined the effects of sxgliptin nd sitgliptin on glycemic control in streptozotocin Improvement of glycemic control in streptozotocininduced dietic rts y Atlntic slmon skin geltin hydrolyste s the dipeptidyl-peptidse IV inhiitor C.H. Hsieh, T.Y. Wng, C.C. Hung, c M.C. Chen, c nd K.C. Hsu *c In our previous study, Atlntic slmon skin geltin hydrolysed with flvourzyme possessed 42.5% dipeptidyl-peptidse (DPP)-IV inhiitory ctivity t the concentrtion of 5 mg/ml. The orl dministrtion of hydrolyste (FSGH) t single dose of 3 mg/d in streptozotocin (STZ)-induced dietic rts for 5 weeks ws evluted for its ntidietic effect. During the 5- week experiment, ody weight ws incresed, food nd wter intke were reduced y FSGH in dietic rts. The dily dministrtion of FSGH for 5 weeks ws effective to lower the lood glucose levels of dietic rts during orl glucose tolernce test (OGTT). After 5-week tretment, plsm DPP-IV ctivity ws inhiited; plsm ctivity glucgon-like peptide-1 (GLP-1), insulin nd insulin-to-glucgon rtio were incresed y FSGH in dietic rts. The results indicte tht FSGH hs the function of inhiiting GLP-1 degrdtion y DPP-IV, resulting in the enhncement of insulin secretion nd improvement of glycemic control in STZinduced dietic rts (STZ)-induced type 2 dietic mice for 45 dys 8. The results showed tht the oth DPP-IV inhiitors ttenuted the increse in wter intke, nd slightly ut significntly improved glycemic control in STZ-treted mice. In ddition, improved β- cell mss nd morphology were lso oserved. There were still some niml nd clinicl studies tht evluted the ntidietic efficcy of DPP-IV inhiitors, e.g. sitgliptin, vildgliptin nd ASP8497 5,6,11. These studies hve demonstrted tht DPP-IV inhiitors could improve the glycemic control minly ttriuted to the plsm DPP-IV inhiition, insulin secretion, ctive GLP- 1 level elevtion, nd glucgon suppression. However, the ntidietic effects of DPP-IV inhiitory peptides were evluted with limited niml nd clinicl studies, nd these studies were lmost investigted with the cute effects. Orl dministrtion of trypsin-treted β- lctogloulin (single dose of 3 mg/kg) or sitgliptin phosphte hydrte (3 mg/kg) 3 min prior to n orl glucose tolernce test (OGTT) significntly lowered lood glucose levels in C57BL/6 mice 12. A peptide, LPQNIPPL, isolted from goud-type cheese showed the in vitro IC 5 vlue of 46 µm ginst DPP-IV nd ws used to exmine its efficcy in vivo in rts 13. The LPQNIPPL-dministered (single dose of 3 mg/kg) group ws oserved to hve significntly lower plsm glucose level ut not to show differences in plsm insulin concentrtion s compred to control group during n OGTT. A previous study reported tht dministrtion of zein hydrolyste (ZeinH; single dose of 5 mg), which induced GLP-1 secretion in mice, resulted in 2.4-fold increse in plsm insulin concentrtion nd 23% decrese in plsm glucose level reltive to the control (met hydrolyste) during the ip glucose tolernce test (IPGTT) in rts 14. In ddition, the Accepted Mnuscript This journl is The Royl Society of Chemistry 13 J. Nme., 13,, 1-3 1

3 Pge 2 of 6 dministrtion of ZeinH incresed ctive GLP-1 level nd decresed plsm DPP-IV ctivity y -26% from sl levels, nd therefore, tht would led to the enhncement of insulin secretion nd the prevention of hyperglycemi in rts. Our previous study hs shown the Atlntic slmon skin geltin hydrolyste possessed gret in vitro DPP-IV inhiitory ctivity 15, nd two peptides within the hydrolyste were identified s Gly-Pro-Al-Glu (372.4 D) nd Gly-Pro-Gly-Al (3.4 D) with their IC 5 vlues ginst DPP-IV of 49.6 nd 41.9 µm. However, the in vivo ntidietic effect of the hydrolyste hs not een evluted. Moreover, the DPP-IV inhiitory peptides were rrely studied y chronic in vivo niml studies. The im of this study ws to exmine the ntidietic effects of DPP-IV inhiitory peptides from Atlntic slmon skin geltin on STZ-induced dietic rts y chronic study. This expected to give insight into the possile utiliztion of Atlntic slmon skin s potentil source in the tretment of type 2 dietes. Results nd discussion Body weight, food, wter intke nd nonfsting lood glucose At the eginning of the experiment (week ), ody weight of the rts in norml group ws higher thn tht in oth DM groups (Fig. 1A). During the 5-week test period, weight gin ws oserved in ll nimls, nd tht of the rts in DM group ws fewer thn those in norml nd DM+FSGH groups. It hs een reported tht nimls receiving STZ susequently showed n initil weight reduction, followed y slower weight gin compred with STZ- nd non-stztreted nimls 8. Food intke, s expected, ws enhnced in DM group rts, ut decresed in DM+FSGH group rts to the levels similr to norml group (Fig. 1B). During the 5-week period, food intke of the rts in DM group incresed rpidly since the third week nd reched pproximtely 4% elevtion t the end of the test. In ddition, there were no significnt differences (P>.5) in food intke etween the rts in norml nd DM+FSGH group, while food intke incresed less thn % during the test period. Wter intke significntly incresed in DM group rts compred with DM+FSGH group rts in week 2 to 5, while during the test period, wter intke showed insignificnt differences (P>.5) etween norml nd DM+FSGH group rts (Fig. 1C). During the 5-week chronic dily dosing study, FSGH significntly decresed nonfsting lood glucose levels from 243 to 178 mg/dl in DM rts (Fig. 1D); while the nonfsting lood glucose levels in DM group rts grdully incresed to 3 mg/dl. A previous study showed tht ASP8497, DPP-IV inhiitor, significntly decresed nonfsting lood glucose nd HA 1c of STZ-induced mildly dietic mice fter the 4-week chronic experiment 11. The result indicted tht FSGH hs the hypoglycemic effect on DM rts. Orl glucose tolernce test At the end of the experiment, OGTT lood glucose responses of the rts in ll groups were shown in Fig. 2A, nd the re under curve (AUC) ws shown in Fig. 2B. The lood glucose levels during OGTT test of DM rts were significntly higher thn the rts in oth norml nd DM+FSGH groups (P<.5). FSGH ws potent to lower the lood glucose levels of dietic rts to less thn mg/dl during OGTT. As the results of the plsm glucose AUC, FSGH significntly improved the lood glucose levels of dietic rts fter 5-week dministrtion (P<.5); menwhile, the glucose AUC of the rts in norml nd DM+FSGH groups ws not significntly different (P>.5). The result ws consistent with the previous study, which reported tht the cheese peptide LPQNIPPL showed hypoglycemic effect on the femle SD rt model 13. A previous study hs reported tht the orl nd single dministrtion of zein hydrolystes significntly lowered glucose levels of dietic rts to round 5% of those of dietic control rts 14. Therefore, the dministrtion of FSGH for 5 weeks is effective for glycemic control of dietic rts. Body weight (g) Wter intke (ml/d) 5 5 A B C Time (weeks) Norml DM DM+FSGH Food intke (g/d) Blood glucose (mg/dl) D Time (weeks) Fig. 1 Weekly verge (A) ody weight, (B) food intke, (C) wter intke, nd (D) nonfsting lood glucose of dietic rts treted with FSGH during 5-week experiment. (n = 12/group) Plsm DPP-IV ctivity The effect of dministrtion of FSGH fter 5 weeks on the plsm DPP-IV ctivity in dietic rts ws shown in Fig. 3. The plsm DPP-IV ctivity of dietic control rts ws 115.5% nd significntly higher thn those of the rts in norml nd DM+FSGH groups (P<.5). The DPP-IV ctivity of dietic rts dministrted with FSGH ws only 82.6%, which showed lower levels thn norml rts (P<.5). The result showed tht FSGH could reduce the plsm DPP-IV ctivity in order to improve the glycemic control in dietic rts. A previous study hs indicted tht DPP-IV inhiitor, NVP- DPP728, orlly dministrted 1 min efore the orl glucose chllenge significntly nd potently inhiited plsm DPP-IV ctivity throughout the OGTT in oese f/f rts 1. For the long term (up to 5 weeks) of DPP-IV inhiitor, sitgliptin, dministrtion in STZ-induced dietic mouse, the plsm DPP-IV ctivity ws gretly inhiited y pproximtely 5%, which strted from the next dy of dministrtion of sitgliptin nd lsted until 5 weeks 6. Plsm totl nd ctive GLP-1 levels After 5-week experiment, the plsm totl GLP-1 level of norml rts ws pm, which ws significntly higher thn those (16.22 nd pm) of the rts in DM nd DM+FSGH groups (P<.5) (Fig. 4A). Menwhile, the totl GLP-1 levels Accepted Mnuscript 2 J. Nme., 12,, 1-3 This journl is The Royl Society of Chemistry 12

4 Pge 3 of 6 of oth DM nd DM+FSGH group rts were insignificntly different (P>.5). The plsm ctive GLP-1 level of DM+FSGH group rts ws significntly higher thn those of the dietic control rts nd norml rts (P<.5) (Fig. 4B). Moreover, the GLP-1 level of the dietic control rts ws similr to tht of norml rts without significnt differences (P>.5). Some studies indicted tht the dily dministrtion of DPP-IV inhiitors (sitgliptin, ASP8497 nd vildgliptin) for up to 1 month resulted in significnt increses in plsm levels of ctive GLP-1 in STZ-induced dietic mice nd resulted in the improvement of glycemic control 6,11. Previous studies hve shown tht zein hydrolyste cn increse GLP-1 secretion in oth GLUTg cells nd norml SD rts models 14,16. However, in the present study, FSGH cn increse ctive GLP-1 levels ut not induce GLP-1 secretion in dietic rts. In nother study, the DPP-IV inhiitor vline pyrrolidide hd no significnt effect on GLP-1 secretion of the STZ-induced dietic minipigs, while tht resulted in n increse in circulting levels of ctive GLP In the similr dietic niml models induced y STZ presented in this nd previous studies, DPP-IV inhiitors showed the protective effects on intct, ctive GLP-1 from the degrdtion y DPP-IV 6,11,17, ut their effects on the increse of GLP-1 secretion were not oserved. Plsm glucose AUC (mg*min/dl) Blood glucose (mg/dl) A B Time (min) Norml DM Norml DM DM+FSGH DM+FSGH Fig. 2 (A) The lood glucose levels nd (B) AUC -18 vlues during orl glucose tolernce test of dietic rts treted with FSGH fter 5-week experiment. Brs with different letters re significntly different t P<.5. (n = 12/group). Plsm DPP-IV ctivity (%) Norml DM DM+FSGH Fig. 3 Effect of dily dministrtion of FSGH on plsm DPP- IV ctivity of STZ-induced dietic rts. Brs with different letters re significntly different t P<.5. (n = 12/group) Plsm totl GLP-1 (pm) Plsm ctive GLP-1 (pm) A B Fig. 4 Effect of dily dministrtion of FSGH on plsm (A) totl nd (B) ctive GLP-1 levels of STZ-induced dietic rts. Brs with different letters re significntly different t P<.5. (n = 12/group) c Norml DM DM+FSGH Accepted Mnuscript This journl is The Royl Society of Chemistry 12 J. Nme., 12,, 1-3 3

5 Pge 4 of 6 Plsm insulin nd glucgon levels The effect of the dily dministrtion of FSGH for 5 weeks on the plsm insulin level in dietic rts ws shown in Fig. 5. The insulin level of norml rts ws out 2.25 µg/l nd higher thn those of dietic rts in the other two groups (P<.5). The dietic rts dministrted with FSGH showed their plsm insulin level over 1.5 µg/l nd significntly higher (P<.5) thn the dietic control rts (Fig. 5). The results showed tht the long-term dministrtion of DPP-IV inhiitory peptides, FSGH, ws potent to improve the insulin secretion in dietic rts. Streptozotocin my induce the loss of pncretic β-cells nd their functionl defects nd therefore result in the reduction of postprndil insulin secretion nd led to hyperglycemi 11. The dily dministrtion of DPP-IV inhiitors, ASP8497 nd vildgliptin (3 mg/kg) in STZ-NA-induced dietic mice for 4 weeks, the glucose-dependent insulin secretion nd ccompnying lood glucose-lowering effects were identified to led to economiztion of insulin nd increses in pncretic insulin content 11. Additionlly, it ws shown tht long-term (4 to 7-week) tretment of STZ-induced dietic rts with DPP-IV inhiitors sitgliptin nd isoleucine thizolodide resulted in reduced lood glucose levels nd increses in pncretic insulin content nd the numer of smll islets 6, 18. Insulin (ug/l) c Norml DM DM+FSGH Fig. 5 Effect of dily dministrtion of FSGH on plsm insulin levels of STZ-induced dietic rts. Brs with different letters re significntly different t P<.5. (n = 12/group) The dietic control rts showed the lowest plsm insulinto-glucgon rtio in the present study; menwhile, the norml control rts hd the highest vlue (P<.5) (Fig. 6). The result showed tht the dily dministrtion of FSGH my improve the control of lood glucose levels of STZ-induced dietic rts. It hs een reported tht the dietic ptients hd reltively high glucgon level tht resulted in glycogenlysis, gluconeogenesis nd then led to hyperglycemi 19. The DPP-IV inhiitor vildgliptin t mg once or twice dily for 4 weeks reduced glucgon response to mixed mel, nd this reduction correlted with the improvement in glucose tolernce 7 ; moreover, study lso showed tht the reduction in glucgon levels y vildgliptin corresponded to inhiition of heptic glucose production 5. The dministrtion of single dose (4 g/kg) of sitgliptin in STZ-induced mice for 4 weeks retined the reltively high plsm insulin-to-glucgon rtio s compred to the dietic control rts, reflecting the control of lood glucose levels 6. The hyperglycemic niml model used in this study is the STZ-NA-induced dult dietic SD rts. As NA is n ntioxidnt which exerts protective effect on the cytotoxic ction of STZ y scvenging free rdicls nd cuses only minor dmge to pncretic β-cell mss producing hyperglycemi. The chemiclly induced dietic niml model shows moderte nd stle non-fsting hyperglycemi with prtil β-cells necrosis nd insulin deficiency. Therefore, this model is found to e n dvntgeous tool for investigtion of insulinotropic gents in the tretment of hyperglycemi 21. However, this niml model shows some shortcomings. First, the niml model hs hyperglycemi which develops primrily y direct cytotoxic ction on the β-cells nd insulin deficiency rther thn consequence of insulin resistnce. Second, this model is disputed to e type 1 or 2 dietes 21,22. In this study, therefore, we hve difficult to determine the effects of the DPP- IV inhiitor on the insulin secretion resulted from the β-cell function when compred with the norml nd dietic groups. From the ove results, the dietic rts showed slight reduction of GLP-1 secretion (Fig. 4A), greter DPP-IV ctivity (Fig. 3) to inctivte GLP-1, ut the similr ctive GLP-1 levels (Fig. 4B) s compred to norml rts. Therefore, we demonstrte tht the hyperglycemi of the dietic rts to e contriuted y the severe insulin deficiency (Fig. 5 nd Fig. 6). The orl dministrtion of FSGH, DPP-IV inhiitor determined in the previous study 15, significntly decresed the DPP-IV ctivity nd incresed the circulting levels of ctive GLP-1 of the dietic rts, lthough the GLP-1 secretion ws not enhnced. The high level of ctive GLP-1 potently stimulted insulin secretion nd then resulted in the improvement of glycemic control. Our previous study hs shown tht the non-dietic rts dministered y the porcine skin geltin hydrolyste s DPP-IV inhiitor did not show ny unexpected chnge, especilly hypoglycemi, during the 42-dy experiment 23. Plsm insulin/glucgon rtio c Norml DM DM+FSGH Fig. 6 Effect of dily dministrtion of FSGH on plsm inslulin-to-glucgon rtio of STZ-induced dietic rts. Brs with different letters re significntly different t P<.5. (n = 12/group) Experimentl Accepted Mnuscript 4 J. Nme., 12,, 1-3 This journl is The Royl Society of Chemistry 12

6 Pge 5 of 6 Mterils nd regents Atlntic slmon (Slmo slr) fish skins, the processing yproducts recovered from fresh skin-off fillets, were supplied y Alion Fisheries Ltd. (Vncouver, BC, Cnd). The fish skins were trnsferred on ice to our lortory, vcuum pcked nd stored t -25 until use. The preprtion of the fish skin geltin hydrolystes ws reported in our previous study 15. Briefly, the extrcted geltin from Atlntic slmon skin ws hydrolysed with flvourzyme with the enzyme/sustrte rtio of 6% for 4 h, nd the hydrolystes were lyophilised nd stored t - until use. Streptozotocin (STZ) nd nicotinmide (NA) were purchsed from Sigm-Aldrich (St. Louis, MO, USA). Other chemicls nd regents used were nlyticl grde nd commercilly ville. Animls Mle Sprgue-Dwley rts (LASCO, Tipei, Tiwn), ged 8 weeks nd weighing etween g were used. Dietes ws induced in overnight (>1 h) fsted rts y single intrperitonel injection of citrte uffer (.1 M, ph 4.5) solution of STZ t dosge of 65 mg/kg ody weight (BW), 15 min fter the i.p. dministrtion of 18 mg/kg BW of NA in norml sline. One week fter the injection of STZ, nimls were considered to e dietic if they hd plsm glucose levels over mg/dl during the orl glucose tolernce test (OGTT). All rts cre nd procedures were pproved y the Institutionl Animl Cre nd Use Committee of Chin Medicl University. Experimentl group nd tretment Animls were divided into 3 groups of 12 rts ech, nd the rts in experimentl groups were dministrted smples y orl gvge. The experimentl period ws 5 weeks. Norml group: norml control rts dministered drinking wter dily; DM group: dietic rts dministered drinking wter dily; DM+FSGH group: dietic rts dministered fish skin geltin hydrolystes (FSGH; 3 mg/dy). Plsm glucose nd orl glucose tolernce test Nonfsting lood glucose levels were mesured in til lood using lood glucose meter (TD-47, Tidoc, New Tipei, Tiwn). The OGTT test ws performed in overnight fsted rts from ll groups, nd the plsm glucose levels were determined t, 3, 6, 9, 1 nd 18 min fter glucose chllenge (2 g/kg). Biochemicl determintions On the morning fter finl dministrtion, the nimls were scrificed y over dose of CO 2. Blood smples under nonfsting conditions were collected in chilled lood vses contining ethylenediminetetrcetic cid (EDTA). Smples were centrifuged (3,g, 15 min) nd stored t -8. Plsm DPP-IV ctivity ws mesured using DPPIV/CD26 ssy kit (Enzo Inc., Frmingdle, NY, USA). Plsm totl nd ctive GLP-1 concentrtions were mesured using glucgon like peptide-1 (totl) RIA kit nd glucgon like peptide-1 (ctive) ELISA kit (Millipore Corp., Billeric, MA, USA), respectively. Plsm insulin concentrtion ws mesured using Mercodi rt insulin kit (Mercodi Inc., Uppsl, Sweden). Plsm glucgon concentrtion ws mesured using glucgon EIA kit (Ynihr Inc., Shizuok, Jpn). Sttisticl nlysis Ech dt point represents the men of three smples ws sujected to nlysis of vrince (ANOVA) followed y Duncn s test, nd the significnce level of P<.5 ws employed. Conclusions FSGH from Atlntic slmon skin geltin hd superior ntidietic effect in STZ-induced dietic rts, including the improvement of glucose tolernce, inhiition of plsm DPP-IV ctivity, elevtion of ctive GLP-1 levels, resulting in the enhncement of insulin secretion, the reduction of glucgon levels nd finlly improvement of glycemic control. This study indictes the Atlntic slmon skin geltin hs the potentil to e functionl food for ntihyperglycemi. Acknowledgements The uthors thnk Dr. Eunice C.Y. Li-Chn (The University of British Columi) for her technicl support s well s Dr. Musleh Uddin (Alion Fisheries Limited) for the kind supply of fish skin. This study ws finncilly supported y Chin Medicl University (Project No. CMU 99-S-37 nd CMU12- ASIA-13). Notes nd references Deprtment of Helth nd Nutrition Biotechnology, Asi University, Tiwn. Division of Endocrine nd Metolism, Chin Medicl University Hospitl, Tiwn c Deprtment of Nutrition, Chin Medicl University, Tiwn. Corresponding uthor: kchsu@mil.cmu.edu.tw Address: Deprtment of Nutrition, Chin Medicl University, 91 Hsueh- Shih Rod, Tichung 442, Tiwn. Tel: ext Fx: Electronic Supplementry Informtion (ESI) ville: [detils of ny supplementry informtion ville should e included here]. See DOI: 1.139/x/ 1 B. Gllwitz, Vsc. Helth Risk Mng., 7, 3, J. A. Pospisilik, S. G. Stfford, H. U. Demuth, C. H. S. McIntosh, R. A. Pederson, Dietes, 2, 51, C. F. Decon, M. A. Nuck, J. Meier, K. Hücking, J. J. Holst, J. Clin. Endocr. Met.,, 85, H. Mitni, M. Tkimoto, T. E. Hughes, M. Kimur, Jpn. J. Phrmcol., 2, 88, B. Bls, M. R. Big, C. Wtson, B. E. Dunning, M. Ligueros- Syln, Y. Wng, Y. L. He, C. Drlnd, D. J. Holst, C. F. Decon, K. Cusi, A. Mri, J. E. Foley, R. A. DeFronzo, J. Clin. Endocr. Met., 7, 92, S. J. Kim, C. Nin, D. J. Doudet, C. H. S. McIntosh, Dietes, 8, 57, Accepted Mnuscript This journl is The Royl Society of Chemistry 12 J. Nme., 12,, 1-3 5

7 Pge 6 of 6 7 A. Mri, W. M., Slls, Y. L. He, C. Wtson, M. Ligueros-Syln, B. E. Dunning, C. F. Decon, J. J. Holst, J. E. Foley, J. Clin. Endocr. Met., 5, 9, S. M. Poucher, S. Cheethm, J. Frncis, B. Zinker, M. Kiry, S. P. Vickers, Dietes Oes. Met., 12, 14, K. Gooen, S. Gräer, Dietes Oes. Met., 12, 14, B. Blkn, L. Kwsnik, R. Miserendino, J. J. Holst, X. Li, Dietologi, 1999, 42, A. Mtsuym-Yokono, A. Thr, R. Nkno, Y. Somey, K. Shirki, M. Hykw, M. Shiski, Metolism, 9, 58, M. Uchid, Y. Ohshi, O. Mogmi, J. Phrmcol. Sci., 11, 117, H. Uenishi, T. Kuki, Y. Seto, A. Serizw, H. Nkjim, Int. Diry J., 12, 22, T. Mochid, T. Hir, H. Hr, Endocrinology, 1, 151, E. C. Y. Li-Chn, S. L. Hung, C. L. Jo, K. P. Ho, K. C. Hsu, J. Agr. Food Chem., 12, 6, T. Hir, T. Mochid, K. Miyshit, H. Hr, Am. J. Physiol-Gstr. L., 9, 297, G663-G M. O. Lrsen, B. Rolin, U. Riel, M. Wilken, C. F. Decon, O. Svendsen, C. F. Gotfredsen, R. D. Crr, Exp. Diesity Res., 3, 4, J. A. Pospisilik, J. Mrtin, T. Doty, J. A. Ehses, N. Pmir, F. C. Lynn, S. Piteu, H. U. Demuth, C. H. McIntosh, R. A. Pederson, Dietes, 3, 52, B. Ahrén, Best Prct. Res. Cl. En., 7, 21, P. Murugn, L. Pri, Life Sci., 6, 79, K. Srinivsn, P. Rmro, Indin J. Med. Res., 7, 125, A. J. F. King, Br. J. Phrmcol., 12, 166, S. L. Hung, C. C. Hung, C. L. Jo, Y. S. Tung, K. C. Hsu, J. Funct. Food, 14, 11, Accepted Mnuscript 6 J. Nme., 12,, 1-3 This journl is The Royl Society of Chemistry 12