The glycemic, insulinemic and plasma amino acid responses to equi-carbohydrate milk meals, a pilot- study of bovine and human milk

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1 Gunnerud et l. Nutrition Journl 2012, 11:83 RESEARCH Open Access The glycemic, insulinemic nd plsm mino cid responses to equi-crohydrte milk mels, pilot- study of ovine nd humn milk Ulrik Gunnerud 1*, Jens J Holst 2, Elin Östmn 1 nd Inger Björck 1 Astrct Bckground: Diry proteins, in prticulr the whey frction, exert insulinogenic properties nd fcilitte glycemic regultion through mechnism involving elevtion of certin plsm mino cids, nd stimultion of incretins. Humn milk is rich in whey protein nd hs not een investigted in this respect. Method: Nine helthy volunteers were served test mels consisting of humn milk, ovine milk, reconstituted ovine whey- or csein protein in rndom order. All test mels contriuted with 25g intrinsic or dded lctose, nd white whet red (WWB) mel ws used s reference, providing 25g strch. Post-prndil levels in plsm of glucose, insulin, incretins nd mino cids were investigted t time intervls for up to 2 h. Results: All test mels elicited lower postprndil lood glucose responses, expressed s iauc min compred with the WWB (P < 0.05). The insulin response ws incresed following ll test mels, lthough only significntly higher fter whey. Plsm mino cids were correlted to insulin nd incretin secretion (iauc 0 60 min) (P 0.05). The lowered glycemi with the test mels (iauc 0 90 min) ws inversely correlted to GLP-1 (iauc 0 30 min) (P 0.05). Conclusion: This study shows tht the glycemic response ws significntly lower following ll milk/milk protein sed test mels, in comprison with WWB. The effect ppers to originte from the protein frction nd erly phse plsm mino cids nd incretins were involved in the insulin secretion. Despite its lower protein content, the humn milk ws potent GLP-1 secretgogue nd showed insulinogenic properties similr to tht seen with reconstituted ovine whey-protein, possily due to the comprtively high proportion of whey in humn milk. Keywords: Amino cids, Bovine milk, GIP, GLP-1, Humn milk, Whey protein Bckground The prevlence of type 2 dietes (T2D) is incresing round the world, with sedentry lifestyles nd indequte dietry hits s the mjor cusing fctors. Epidemiologicl evidence suggests, tht diet chrcterized y low glycemic index (GI), protects ginst the development of T2D [1]. The GI is used to estlish postmel glycemic responses to crohydrte rich mels. Most often the GI correltes with Insulinemic Index (II), mesured similrly from postprndil insulinemic responses. However, milk displys discrepncy, * Correspondence: ulrik.gunnerud@ppliednutrition.lth.se 1 Deprtment of Applied Nutrition nd Food Chemistry, Lund University, P.O. Box 124, , Lund, Sweden Full list of uthor informtion is ville t the end of the rticle showing low GI ut yet high II [2]. Oservtionl studies hve indicted tht diet rich in diry products my protect ginst development of the metolic syndrome nd T2D [3,4]. Additionlly, ovine whey protein stimultes insulin secretion nd fcilittes post-mel glycemic regultion in oth helthy individuls nd in ptients with T2D [5-8]. This my imply tht coingestion of milk, in prticulr the whey frction, with mels my lower glycemic excursions nd therey reduce the glycemic impct of the diet. In previous study, we hve shown tht whey protein resulted in higher insulin response thn cheese (csein) or milk (whey nd csein), respectively [6]. The mjor insulin relesing properties of milk could thus e scried to the whey frction, nd the mechnism 2012 Gunnerud et l.; licensee BioMed Centrl Ltd. This is n Open Access rticle distriuted under the terms of the Cretive Commons Attriution License ( which permits unrestricted use, distriution, nd reproduction in ny medium, provided the originl work is properly cited.

2 Gunnerud et l. Nutrition Journl 2012, 11:83 Pge 2 of 9 ppers to e relted to the specific mino cid pttern ppering in postprndil lood fter whey ingestion [6]. When dministrted with crohydrtes, dietry mino cids stimulte the insulin relese from the pncretic β- cell [9]. Especilly leucine (leu) is known to e strong insulin secretgogue [10]. Consequently, the lowering of glycemi seen when dding whey to crohydrte contining mel cn to lrge extent e mimicked y supplementtion of the mel with specific mino cids [11]. In ddition, whey proteins pper to stimulte the incretin hormones glucose-dependent insulinotropic polypeptide (GIP) nd glucgon-like peptide 1 (GLP-1) [12]. Both GLP-1 s well s GIP hs een identified s strong insulinotropic gents [13]. Furthermore, it hs recently een reported tht dietry rnched chin mino cids (BCAA) fcilittes GLP-1 relese from intestinl-cells [14]. Thus, plsm mino cids per se cn possily cuse n incretin secretion, nd prtly explin the reduction of glycemi seen when dding whey to crohydrte contining mel. The protein concentrtion in humn milk is considerly lower thn in ovine milk (8.5 g/l vs g/l) [15]. Also the composition of the protein frctions is sustntilly different, with humn milk contining lrger frction of whey protein. Consequently, the rtio whey:csein differs etween the two milk types, eing 20:80 for ovine milk [16] nd 50:50 80:20 to humn milk, with somewht different rtios depending on the lctting phse [17]. Bsed on ove, it cn e hypothesized tht humn milk, despite its low protein content, exerts insulinogenic properties due to its high whey protein content. To our knowledge, no dt re ville regrding the postprndil glycemic, insulinemic nd incretin effects of humn milk. The im of the present study ws to investigte the glycemic nd insulinemic effects of humn nd ovine milk in comprison with white whet red (WWB). In ddition, csein nd whey protein frctions from ovine milk were included. For this purpose, humn milk, ovine milk, ovine csein nd whey proteins, respectively, were stndrdized with respect to lctose content nd provided s rekfst drinks to helthy dults. The study ws performed in glycemic index mesuring setting, nd crohydrte equivlent mount of white whet red (WWB) ws therefore used s reference mel. In ddition to lood glucose nd insulin, plsm levels of mino cids nd the incretins GIP nd GLP-1 were mesured in the postprndil phse. Methods Test mels Four test drinks were included in the study; humn nd ovine milk nd reconstituted ovine whey nd csein drinks. The nutritionl compositions of the mels re shown in Tle 1. Humn milk, whey nd csein concentrtes were otined from Arl Foods (Stockholm, Sweden). Bovine milk with 1.5% ft (w/v) ws otined from locl mrket. The humn milk ws skimmed to reduce ft content (1.5% w/v), followed y psteuriztion. According to the mnufcture the whey nd csein proteins were otined y ultrfiltrtion nd memrne filtrtion of skimmed milk. Lctose ws then dded to the protein frctions nd the mixtures were dissolved in wter. Ech of the mels contriuted with 25 g ville crohydrtes, thus the protein content differed significntly etween the ovine milk nd the humn milk mels (16.8 g nd 3.5 g respectively). The reconstituted whey nd csein mels were set to mtch the ovine milk with respect to lctose-, protein nd ft contents. The serving of whey contined 16.2 g of protein nd the csein mel contined 16.8 g protein. All the test drinks were set to mtch the ovine milk in ft content (1.5% w/v). Humn milk, whey nd csein drinks were kept frozen ( 18 C) until use. White whet red (WWB) ws used s reference mel. The WWB ws prepred in home king mchine ccording to Liljeerg nd Björck [18]. Test sujects nd study design Nine helthy, non-smoking, volunteers, (6M: 3F), ged yers, with ody mss index 25.8 ± 3.4 kg/ m 2 (men ± SD) nd without drug tretment prticipted in the study. All sujects hd norml fsting lood glucose levels (4.4 ± 0.05 mmol/l). The mels were served in Tle 1 Nutrient composition nd serving sizes of the test mels WWB 1 Whey 2 Csein 2 Bovine milk 2 Humn milk 2 Strch (g) Lctose (g) Protein (g) Ft (g) Serving quntity (g) Σ Crohydrtes (g) Solid food. 2 Liquid foods.

3 Gunnerud et l. Nutrition Journl 2012, 11:83 Pge 3 of 9 rndom order s rekfst mels t 5 different occsions, t lest 2 dys prt. When rriving t the lortory in the morning (8 m), peripherl ctheter ws inserted into the ntecuitl vein nd fsting lood smple ws drwn. At ll smpling time points, smples were tken in one tue for serum nd one for plsm (EDTA), respectively. The sujects were requested to consume the test nd reference mels stedily over 12 min period. All sujects were conscious of the possiility to withdrw from the study t ny time they desired. The study ws pproved y the Ethics Committee of the Fculty of Medicine t Lund University. Chemicl nlysis Lctose content of the test mels ws nlyzed s glctose nd glucose following enzymtic hydrolysis with β- glctosidse s descried y Nilsson et l. [6].The peptide-ound mino cids of the different food proteins were nlyzed ccordingly Stenerg et l. [19] strting with hydrolysis step. The smples were dissolved in 6 M hydrochloric cid, contining 0.1% phenol nd kept t 110 C for 20 h. As result of the hydrolysis tretment, tryptophn, cysteine nd methionine were lost nd therefore dt for these mino cids re lcking in the results. Furthermore, due to the hydrolysis step, glutmine nd sprgine were converted to glutmic cid nd sprtic cid, respectively. The mino cids were nlyzed using ion exchnge chromtogrphy (Biotronik LC 5001, München, Germny). Stndrd lithium citrte uffers of ph 2.85, 2.89, 3.20, 4.02 nd 3.49 were used to seprte the mino cids. The post-column derivtiztion ws performed with ninhydrin [20]. The crude protein content in ll the mels ws nlyzed y using the Kjeldhl procedure (Kjeltec Auto 1030 Anlyser; Tector Högnäs, Sweden). The strch in the WWB ws determined ccording to the method of Holm et l. [21]. Blood nlysis Venous lood smples were tken t fsting (0) nd, t 7.5, 15, 30, 45, 60, 75, 90, 105 nd 120 min fter the mel were initited, for nlysis of lood glucose, serum insulin nd GIP nd GLP-1 nlysis. Additionlly, plsm ws collected t 0, 7.5, 15, 30, 45 nd 60 min for mino cids nlysis. The plsm tues were llowed to rest for 30 min efore eing centrifuged t 3000 rpm during 6 min. 800 μl plsm ws seprted for free mino cids nlysis nd the remining plsm ws frozen t 20 C efore nlysis of GIP nd GLP-1. Serum tues were centrifuged for 15 min t 4000 rpm nd serum ws seprted nd frozen t 20 C for insulin nlysis. Whole lood glucose concentrtions were determined with glucose oxidse-peroxidse regent. The serum insulin determintion ws performed on n integrted immunossy nlyzer, CODA Open Microplte System (Bio-Rd Lortories, Hercules, USA) using n enzyme immunossy kit (Mercodi Insulin Elis, Mercodi AB, Uppsl, Sweden). Plsm GIP nd GLP-1 concentrtions were mesured fter extrction of plsm with 70% ethnol (y vol, finl concentrtion). For the GIP rdioimmunossy [22], we used the C-terminl directed ntiserum R 65, tht cross-rects fully with humn GIP ut not with so clled GIP 8000, whose chemicl nture nd reltion to GIP secretion is uncertin. Humn GIP nd 125 I humn GIP (70 MBq/nmol) were used for stndrds nd trcers. The concentrtion of plsm GLP-1 ws mesured [23] ginst stndrds of synthetic GLP mide y using ntiserum code no , which is specific for the midted croxyl terminus of GLP-1 nd, therefore, does not rect with GLP-1 contining peptides from the pncres. For oth ssys sensitivity ws <1 pmol/l, intrssy CV <6% t 20 pmol/l, nd recovery of stndrd, dded to plsm efore extrction, 100% when corrected for losses inherent in the plsm extrction procedure. Free mino cids were purified y mixing 200 μl 10% sulphoslic cid with 800 μl plsm to precipitte high moleculr proteins, ccording to Biotronik (München, Germny). The mino cid solutions were frozen t 20 C until nlyzed y n mino cid nlyzer (Biotronik LC 5001, München, Germny) using ion exchnge chromtogrphy s descried ove. Clcultions nd sttisticl methods The results re expressed s men ± SEM. The incrementl res under the curves (iauc) for glucose, insulin, GIP, GLP-1 nd plsm mino cids were clculted for ech test suject nd mel, using trpezoid model (GrphPd Prism, version 4.03). All res elow the seline were excluded from the clcultions nd ech suject ws their own reference. Sttisticl comprisons for iauc-vlues were performed in MINITAB Sttisticl Softwre (relese for windows). Significnces were evluted with the generl liner model (ANOVA), followed y Tukey s multiple comprisons test. Sttisticl significnces were considered for vlues of P < Differences etween the products t different time points were nlyzed y using mixed model (PROC MIXED in SAS relese 9.1; SAS Institute Inc, Cry, NC) with repeted mesures nd n utoregressive covrince structure. When significnt interctions etween tretment nd time were found, Tukey s multiple comprison test were performed for ech time point (MINI- TAB, relese 14.13; Minit Inc). Correltions etween dependent mesures were conducted using Spermn's prtil correltion coefficients controlling for sujects nd corresponding seline vlues (two-tiled test), (SPSS softwre, version 19; SPSS Inc., Chicgo, IL, USA). Due to missing vlues the ovine milk hd to e

4 Gunnerud et l. Nutrition Journl 2012, 11:83 Pge 4 of 9 withdrwn from the GLP-1 nd GIP sttisticl clcultions, nd the mino cid isoleucine (ile) hd to e excluded from the clcultions on free mino cids in plsm. In ddition, there were only eight test sujects on the WWB nd whey, nd seven test sujects on the csein nd the humn milk respectively included in the GIP nd GLP-1 clcultions, due to missing vlues. Results Postprndil lood glucose nd insulin responses The iaucs for plsm glucose nd serum insulin re shown in Tle 2. All test mels elicited lower postprndil lood glucose responses, expressed s iauc min, compred with the reference (WWB) (p < 0.05). A significnt tretment effect (p = ) nd significnt time tretment interction (p = ) ws found for lood glucose concentrtions (Figure 1). The insulin iauc following humn milk, ovine milk nd csein ws similr to tht seen with WWB. In contrst, the whey mel exhiited higher insulin iauc min, thn the WWB (p < 0.05). Additionlly, the whey mel resulted in n elevted insulin response (iauc min) lso when compred with dt for csein nd humn milk. No time tretment interction ws found for insulin responses (Figure 1). Amino cid content in the test nd reference mels The mino cid concentrtions of the different mels re presented in Tle 3. Bovine milk, csein nd whey hve similr ptterns, with higher concentrtions of the BCAA; ile, leu, nd vline (vl) compred to humn milk nd WWB. The whey mel stnds out y higher leu content, nd ovine milk nd csein, respectively, hve slightly higher mounts of vl. In ccordnce to its lower protein content, the humn milk disply consistently lower content of ll nlyzed mino cids compred to the whey, csein nd ovine milk. In comprison to the WWB, the humn milk hs higher concentrtions of vl, leu nd lys, wheres the WWB hve high proline, glycine nd tyrosine (tyr) levels. Postprndil plsm mino cids The 0 60 min iauc for plsm mino cids re displyed in Tle 4. The postprndil responses for vl, leu, lys, serine (ser), lnine nd rginine (rg) were significntly higher following whey, csein nd ovine milk compred to the WWB. Furthermore, csein nd whey resulted in significntly higher iauc for rg nd ser compred to the humn milk. The tyr response ws significntly higher fter the whey protein nd the ovine milk, respectively, compred with the WWB nd the humn milk. The whey nd csein mels lso elicited significntly higher plsm iauc for threonine thn ll the other mels. There were no differences etween the humn milk nd the WWB in ny of the nlyzed mino cids in postprndil plsm. Postprndil GLP-1 nd GIP responses The iaucs min for GLP-1 nd GIP re shown in Tle 2. The whey mel resulted in significntly higher iauc min for oth GIP s well s GLP-1, in comprison to ll the other mels. Also in the cse of the iaucs 0 45, 0 60 nd 0 90 min, respectively the whey mel elicited higher increment for oth GIP nd GLP-1 compred with WWB (p 0.05). In ddition, the humn milk elicited significntly higher erly postprndil GLP-1 response, expressed s iaucs 0 30 nd 0 45 min, compred to the WWB (p 0.05) (erly iauc not displyed). Incretin responses following the csein mel showed no differences compred to the WWB. Significnt differences etween tretments over the entire time course (p 0.001) nd significnt time tretment interctions (p 0.001) were found for oth GLP-1 nd GIP responses (Figure 2). Correltions etween prmeters Correltions etween plsm mino cids, serum insulin nd plsm incretins re displyed in Tle 5. Positive correltions were oserved etween the plsm mino cids leu, lys, vl nd thr iauc 0 60 min nd the corresponding iauc for serum insulin, nd plsm incretin Tle 2 Postprndil incrementl res under the curve (iauc min) for plsm glucose, serum insulin, plsm GLP-1 nd GIP Glucose 1 Insulin 1 GLP-1 2 GIP 2 mmol min % 4 nmol min % 4 pmol min % 4 pmol min % 4 WWB 96.8 ± ± ± ± Humn milk 48.0 ± ± ± ± Whey 54.1 ± ± ± ± Csein 35.1 ± ± ± ± Bovine milk 40.6 ± ± Vlues re mens ± SEM. Vlues within the sme column not shring sme letters re significntly different (P < 0.05). 1 n = 9 helthy sujects. 2 n = 8 helthy sujects (WWB, whey) nd n = 7 (csein, humn milk). 4 Chnge in postprndil response s percentge of the WWB reference mel.

5 Gunnerud et l. Nutrition Journl 2012, 11:83 Pge 5 of 9 Blood glucose (mmol/l) A WWB Reference Bovine milk Csein Humn milk Whey Serum insulin (nmol/l) B C GI II WWB Humn Bovine Whey Csein Time (min) Time (min) Figure 1 Incrementl chnges in plsm glucose nd serum insulin. Men incrementl chnges (Δ) in plsm glucose (A) nd serum insulin (B) in response to equl mounts of crohydrte. In (C) the GI nd II (iauc) re displyed. For plsm glucose significnt tretment effect (p < ) nd time tretment interction (p < ) were found t given time. Vlues with different lowercse letters re significntly different, p 0.05 (Tukey s test). n = 9 helthy suject. responses. The individul plsm mino cids (leu, lys, vl nd thr) iauc 0 45 min correlted inversely to glucose response t iauc 0-60min (dt not showed). In ddition, erly phse GLP-1 response, expressed s iauc 0 30 min, correlted positively to the erly phse insulin response (iauc 0 30 min; r = 0.688, p < 0.001). Moreover, did the erly phse GIP nd GLP-1, expressed s iaucs 0 15 min, correlte negtively with lood glucose responses iauc 0 90 min (r = 0.419, p = 0.040; r = 0.374, p = 0.012, respectively). Tle 3 Content of mino cids in the different mels Mel Amino cid WWB Whey Csein Bovine milk Humn milk mg/serving Al Arg Asp Glu Gly His Ile Leu Lys Phe Pro Ser Thr Tyr Vl Discussion To the est of our knowledge, this is the first study where humn milk is evluted with respect to glycemic nd insulinemic effects. The study ws performed in helthy dults with GI setting, thus post prndil glycemic nd hormonl responses to the humn nd ovine milk were compred to n equicrohydrte mount of WWB. In the present study, we found tht humn milk displyed insulinogenic properties nd resulted in postprndil glycemic response (GI 57) tht ws in the sme rnge s tht to ovine milk (GI 43) or the whey nd csein frctions, respectively (GI 61 nd 44). The humn milk showed the lowest insulin response (iauc min) in comprison to ll the other mels, thus indicting tht humn milk disply superior insulin economy. Worth emphsizing is tht the protein content of the humn milk mel ws mrkedly lower thn tht of ovine milk, whey nd csein mels. According to our nlysis, the humn milk mel contined 3.5g protein s clculted from nitrogen nlysis using 6.38 s conversion fctor. However, it hs een hs reported tht the true protein content of humn milk is overestimted from nitrogen nlysis due to its high non protein nitrogen (NPN) content (20-25% of totl nitrogen) [16,17]. Consequently, the protein mount in the humn milk, used in this study, my hve een overestimted. Although the whey:csein rtio of the humn nd ovine milk in this study ws not nlyzed, it hs een reported to rnge from 50:50 80:20 in humn milk depending on the lcttion period [17], compred to pproximtely 20:80 in commercil ovine milk [16]. Our results thus indicte tht humn milk my e more insulinogenic per unit protein compred to ovine milk, nd the

6 Gunnerud et l. Nutrition Journl 2012, 11:83 Pge 6 of 9 Tle 4 Postprndil res under the curve (iauc 0 60 min) for the different plsm mino cids Mel Amino cid WWB Whey Csein Bovine milk Humn milk (mmol min/l) Al 0.4 ± ± ± ± ± 0.5 Arg 0.2 ± ± ± ± ± 0.1 Glu 0.9 ± ± ± ± ± 0.5 Gly 0.3 ± ± ± ± ± 0.1 Hist 0.3 ± ± ± ± ± 0.4 Leu 0.3 ± ± 0.5 c 2.7 ± ± ± 0.2 Lys 0.5 ± ± ± ± ± 0.3 Phe 0.5 ± ± ± ± ± 0.1 Pro 2.4 ± ± ± ± ± 0.4 Ser 0.1 ± ± 0.2 c 0.9 ± 0.2 c 0.8 ± 0.2 c 0.3 ± 0.1 Thr 0.2 ± ± ± ± ± 0.2 Tyr 0.2 ± ± ± ± ± 0.1 Vl 0.6 ± ± ± ± ± 0.2 Vlues re mens ± SEM; n = 9. Vlues in the sme column with different letters re significntly different, p < 0.05 (ANOVA followed y Tukey s test). higher proportion of whey protein my e contriuting fctor. All the test products in our study yielded higher postprndil plsm mino cid responses compred to the WWB reference mel, lthough not reching significnce for ll products. The ovine milk nd the reconstituted whey nd csein mels were especilly prone to ffect responses of the BCAA; vl nd leu, nd lso lys nd thr. We lso noticed tendency to rise in these mino cids following humn milk in comprison to the WWB. We recently showed in vitro tht the mino cids ile, leu, vl, lys nd thr induce strong insulin secreting properties in mouse pncretic islets [24]. In prticulr, leu is recognized s potent insulin secretgogue [25,26]. Moreover it hs een reported tht lso turine, the second most common mino cid nd the lrgest prt of the NPN frction in humn milk [27,28], my e involved in insulin secretion [29,30]. In the present study, we lso oserved positive correltions etween individul plsm mino cids nd serum insulin nd plsm incretin secretion in the postprndil phse (iauc 0 60 min) s well s negtive correltions to the glycemic response (iauc 0-60min). Altogether this suggests tht the mino cids ply n importnt role in the insulinogenic properties of the milk proteins nd contriute to lowered postprndil glycemi. Thus, it cn e suggested tht mino cids ppering in plsm following milk ingestion, my ffect insulin secretion in two wys; directly y cting on the pncretic β-cells nd indirectly y promoting incretin relese. We thus found tht humn milk, ovine whey nd csein respectively, ffect relese of GLP-1, with the humn milk resulting in significntly higher erly response compred to the WWB mel. In reltion to the humn milk, the whey GLP-1 (pmol/l) , c c A,, Time (min) WWB Reference Csein Humn milk Whey GIP (pmol/l) B 15 5, Time (min) WWB Reference Csein Humn milk Whey Figure 2 Incrementl chnges in plsm GIP nd GLP-1. Men incrementl chnges (Δ) in plsm GLP-1 (A) nd GIP (B) in response to equl mounts of crohydrte. Significnt tretment effects (p < 0.001) s well s time tretment interctions (p < 0.001) were found for oth GLP-1 nd GIP. Vlues with different letters re significntly different, p < 0.05 (Tukey s test). n = 8 (WWB, whey) nd n = 7 (humn milk, csein).

7 Gunnerud et l. Nutrition Journl 2012, 11:83 Pge 7 of 9 Tle 5 Correltions etween serum insulin, GIP nd GLP-1 nd plsm mino cids Insulin GLP-1 GIP Leu r p < Lys r p <0.001 <0.001 Thr r p < Vl r p Spermn s correltion coefficients nd p-vlues for the reltions etween increments in plsm mino cid concentrtions nd the corresponding serum insulin, plsm GLP-1 nd GIP increment, respectively (iauc 0 60 min). mel yielded higher GLP-1 response. However, considering the fct tht the protein content in the humn milk mel constitutes only out 22% of tht in the ovine whey mel, the erly GLP-1 response (iauc 0 30 min) still reched more thn 50% of tht seen following whey. Thus, it could e suggested tht humn milk is prone to stimulte GLP-1. It remins to e shown if the GLP-1 response is solely relted to the proteins in the humn milk, or if other ioctive components my lso e involved. Strong positive correltions were found etween the erly GLP-1 response (iauc 0 15 min) nd postprndil insulin relese up to 30 min, s well s negtive correltions to lood glucose response (iauc 0 90 min), n indiction tht erly GLP-1 relese my e importnt for the modultion of glycemi following oth humn nd ovine milk. In support of such n opinion, we recently oserved tht ingestion of whey protein nd free mino cid cused n erly GLP-1 secretion tht reduced postprndil glycemi in sence of hyper-insulinemic pek [31]. In ddition to GLP-1, the humn milk lso resulted in smll elevtion of erly phse GIP tht ws significntly higher thn fter the WWB reference mel t 7.5 min. GIP my e present in humn milk [32], nd the response we found might result from higher intke of this incretin, nd could possily ttenute the postprndil insulin response nd fcilitte glycemic regultion. Although, little is known out to wht extent ingested GIP cn mintin ctivity following exposure to the cidic environment in the stomch, it could e hypothesized tht ingestion of intrinsic GIP could possily contriute to the incretin effect of humn milk. In previous studies of incretin responses we hve preferentilly seen increse in GIP fter whey ingestion [5]. Thus, it is interesting tht the whey mel, s well s the humn milk mel, in this study significntly incresed oth GLP-1 nd GIP response compred to ll the other mels. GLP- 1 secretion following whey ingestion hs previously een reported y Hll et l. [33]. However, in tht study the test mels contined 38E% of ft, which my hve influenced GLP-1 secretion. Both GLP-1 nd GIP hve een identified s strong insulinotropic gents [13,34], nd relese of oth these incretins fter whey ingestion my dd to its insulinogenic properties. Interestingly, it ws recently reported tht whey protein inhiits dipeptidyl peptidse IV (DPP-4), n enzyme tht inctivtes GLP-1 [35]. Consequently, whey protein my oth induce GLP- 1 secretion s well s prolong the ctivity of this incretin. In contrst to whey, the csein mel resulted in smll incrementl insulin response nd hd the lowest glycemic response compred to the other test mels. This differences in glycemic nd insulinemic responses when, compring the csein nd whey mel, is in contrst with oservtions y Hll et l. [33], who found no differences on neither postprndil glucose nor insulin responses etween these proteins. However, in ccordnce with the present study, they oserved tht whey protein, in comprison to csein, incresed plsm incretins, with higher levels of oth GLP-1 nd GIP [33]. There were some limittions of the present study. All the test mels resulted in lower lood glucose increments compred to the WWB reference mel. It should, however, e noted tht lctose ws the crohydrte source in ll the test mels, wheres the reference WWB mel contined strch. In previous studies, we hve shown tht lctose hs lower glycemic index (GI 68) nd insulinemic index (II 50) thn the strchy WWB (GI 100 nd II 100) [2]. Consequently, the comprtively low glycemic properties seen with ll the present test mels could prtly e explined y the lctose per se. The numer of test suject is slightly lower (n = 9) thn recommended for stndrdized glycemic index determintions (n = 10) [36]. Also, lthough the test sujects were used to regulr Swedish food hits where milk consumption is frequent, differences in lctse levels might ffect glycemi to milk. Conclusion The present study showed tht humn milk s well s ovine milk nd whey nd csein protein lowered the glycemi compred to the WWB reference mel. We lso demonstrted tht the humn milk exerted insulinogenic properties nd the effect ppered to originte from the protein frction. Consequently, ll the test mels incresed plsm mino cid levels, nd the mino cids correlted positively with oth insulin secretion nd incretin responses s well s inversely to the glycemic response. Interestingly, the humn milk ppered to e prticulrly potent GLP-1 secretgogue. The

8 Gunnerud et l. Nutrition Journl 2012, 11:83 Pge 8 of 9 reltive potency of humn milk to stimulte incretins is noteworthy nd the impct of incretins for metolism nd ppetite regultion is n interesting field for further reserch nd should e tken into considertion when producing y formuls sed on ovine milk. Arevitions iauc: Incrementl re under the curve; BCAA: Brnched chin mino cids; GI: Glycemic index; GIP: Glucose-dependent insulinotropic polypeptide; GLP- 1: Glucgon-like peptide 1; II: Insulinemic index; NPN: Non-protein nitrogen; T2D: Type 2 dietes; WWB: White whet red. Competing interests The uthors declre tht they hve no competing interest. Authors contriutions UG ws responsile for smple collection nd nlysis of the dt, sttisticl nlysis nd for writing the pper. JJH ws responsile for the incretin nlysis. EÖ ws involved in interprettion of dt nd in writing the pper. 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9 Gunnerud et l. Nutrition Journl 2012, 11:83 Pge 9 of Gunnrsson PT, Winzell MS, Decon CF, Lrsen MO, Jelic K, Crr RD, Ahren B: Glucose-induced incretin hormone relese nd inctivtion re differently modulted y orl ft nd protein in mice. Endocrinology 2006, 147: Brouns F, Bjorck I, Fryn KN, Gis AL, Lng V, Slm G, Wolever TM: Glycemic index methodology. Nutr Res Rev 2005, 18: doi: / Cite this rticle s: Gunnerud et l.: The glycemic, insulinemic nd plsm mino cid responses to equi-crohydrte milk mels, pilotstudy of ovine nd humn milk. Nutrition Journl :83. Sumit your next mnuscript to BioMed Centrl nd tke full dvntge of: Convenient online sumission Thorough peer review No spce constrints or color figure chrges Immedite puliction on cceptnce Inclusion in PuMed, CAS, Scopus nd Google Scholr Reserch which is freely ville for redistriution Sumit your mnuscript t