Leucine supplementation differentially enhances pancreatic cancer growth in lean and overweight mice

Transcription

1 Liu et l. Cncer & Metolism 214, 2:6 Cncer & Metolism RESEARCH Open Access Leucine supplementtion differentilly enhnces pncretic cncer growth in len nd overweight mice Kristyn A Liu 1, Lur M Lshinger 1, Audrey J Rsmussen 1 nd Stephen D Hursting 1,2* Astrct Bckground: The risk of pncretic cncer, the 4th dedliest cncer for oth men nd women in the United Sttes, is incresed y oesity. Clorie restriction () is well-known dietry regimen tht prevents or reverses oesity nd suppresses tumorigenesis in vriety of niml models, t lest in prt vi inhiition of mmmlin trget of rpmycin (mtor) signling. Brnched-chin mino cids (BCAA), especilly leucine, ctivte mtor nd enhnce growth nd prolifertion of myocytes nd epithelil cells, which is why leucine is populr supplement mong thletes. Leucine is lso incresingly eing used s tretment for pncretic cncer cchexi, ut the effects of leucine supplementtion on pncretic tumor growth hve not een elucidted. Results: Supplementtion with leucine incresed pncretic tumor growth in oth len (14 ± 17 mm 3 versus 46 ± 13 mm 3 ; P <.5) nd overweight (367 ± 45 mm 3 versus 23 ± 39 mm 3 ; P <.1) mice, ut tumor enhncement ws ssocited with different iologicl outcomes depending on the diet. In the len mice, leucine incresed phosphoryltion of mtor nd downstrem effector S6 riosoml protein, ut in the overweight mice, leucine reduced glucose clernce nd thus incresed the mount of circulting glucose ville to the tumor. Conclusions: These findings show tht leucine supplementtion enhnces tumor growth in oth len nd overweight mice through diet-dependent effects in murine model of pncretic cncer, suggesting cution ginst the clinicl use of leucine supplementtion for the purposes of skeletl muscle enhncement in cchectic ptients. Keywords: Pncretic cncer, Leucine supplementtion, Clorie restriction, mtor, Glucose metolism Bckground Effective prevention nd tretment strtegies re urgently needed for pncretic cncer, the 4th leding cuse of cncer-relted deth in oth men nd women in the United Sttes [1]. Less thn 15% of pncretic cncer ptients hve loclized disese menle to curtive resection, nd the overll 5-yer survivl rte in ffected ptients is less thn 5% [2]. Oesity is n estlished pncretic cncer risk nd progression fctor in humns nd niml models [3-5]. In contrst, clorie restriction () prevents or reverses oesity nd relted metolic * Correspondence: shursting@ustin.utexs.edu Equl contriutors 1 Deprtment of Nutritionl Sciences, University of Texs t Austin, Austin, TX 78723, USA 2 Deprtment of Moleculr Crcinogenesis, University of Texs M.D. Anderson Cncer Center, 188 Prk Rod 1c, Smithville, TX 78957, USA perturtions nd pncretic tumor development nd/or progression in experimentl models [6-11]; the impct of on humn pncretic cncer hs not een well studied. results in negtive energy lnce stte nd exerts its ntitumor effects, t lest in prt, through decresed mmmlin trget of rpmycin (mtor) signling in mny epithelil tissues [7-9,11,12]. mtor cts s nutrient sensor tht regultes protein synthesis, cell survivl, nd prolifertion in response to growth fctor levels, nutrient vilility, nd intrcellulr energy sttus. We hve previously estlished tht rpmycin ( selective mtor inhiitor), nd metformin (n indirect inhiitor of mtor signling through its effects on gluconeogenesis nd ssocited ctivtion of AMPK-regulted signls), prtilly mimic the tumor inhiitory effects of on trnsplnted pncretic tumor growth [13]. 214 Liu 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 credited. The Cretive Commons Pulic Domin Dediction wiver ( pplies to the dt mde ville in this rticle, unless otherwise stted.

2 Liu et l. Cncer & Metolism 214, 2:6 Pge 2 of 12 Brnched-chin mino cids (BCAAs), which ccount for over 2% of totl dietry protein intke, re known ctivtors of the mtor pthwy in muscle nd epithelil tissues [14-16]. Of the three BCAAs, leucine exerts the most potent effect on mtor ctivtion nd enhncement of protein synthesis in vrious tissues, including skeletl muscle [17,18]. Athletes commonly use leucine supplementtion to ctivte mtor-regulted protein synthesis nd ccelerte muscle repir nd regenertion fter injuries or intense outs of exercise [19]. Leucine supplementtion is lso incresingly eing recommended to reduce the muscle wsting tht occurs with cncer cchexi [2]. Cchexi is chrcterized y involuntry weight loss nd muscle wsting, is ssocited with incresed moridity nd mortlity, nd frequently occurs in pncretic cncer ptients [21]. Incresed muscle protein synthesis in response to leucine-induced mtor ctivtion hs een shown to inhiit muscle wsting in mouse models of cncer cchexi nd in cncer ptients [22-25]. However, the rtes of protein synthesis increse to much greter extent in tumors thn in muscle [24], suggesting tht while leucine supplementtion my protect ginst cncer-ssocited cchexi, it my lso enhnce the progression of the cncer. Unfortuntely, studies of the effects of leucine supplementtion on cncer re limited. Long-term leucine supplementtion (2% of diet, w/w) promoted ldder cncer development in rts treted with known ldder crcinogen [26,27], ut no studies hve connected leucine supplementtion with tumor growth. In the present study, we tested the effect of leucine supplementtion on trnsplnted Pnc2 mouse pncretic cncer growth nd mtor signling in the context of len mice (fed diet regimen) or overweight mice (fed high clorie control diet regimen). Our findings suggest tht leucine enhnces pncretic tumor progression in len nd overweight mice, nd the underlying mechnisms my differ y weight sttus. Methods Mice nd dietry interventions All experiments were conducted under protocol pproved y the Institutionl Animl Cre nd Use Committee t the University of Texs t Austin. Eighty-eight mle C57BL/6 mice were otined from Chrles River Breeding Lortories (Wilmington, MA, USA) t 6 to 8 weeks of ge, nd upon rrivl were singly-housed in semirrier fcility t the University of Texs t Austin Animl Resource Center nd fed chow diet during one-week cclimtion period. Mice were then rndomized to receive one of four diets for 27 weeks: (i) AIN-76A control diet consumed d liitum (control, n = 22); (ii) 3% diet (, n = 22); (iii) control diet with leucine supplementtion (5% of diet, w/ w) consumed d liitum (control+leu, n=22); or (iv) 3% diet with leucine supplementtion ( + LEU, n = 22). The AIN-76A control diet, when consumed d liitum, results in n overweight phenotype chrcterized y stedy weight gin, while the results in len phenotype chrcterized y weight mintennce [28,29]. Both diets were dministered s dily liquot providing 7% of the totl energy ut 1% of the vitmins, minerls, mino cids nd ftty cids consumed y the controls. Leucine ws purchsed from AIDP, Inc. (City of Industry, CA, USA) nd ws incorported into the AIN- 76A diet premix to provide 5 g/kg feed, or 5% dietry leucine supplementtion. This dose of leucine is commonly used in niml studies of muscle regenertion [3,31]. All diets were purchsed from Reserch Diets (New Brunswick, NJ, USA). Energy intkes nd ody weights for ech mouse were recorded weekly for 21 weeks, nd then glucose tolernce tests (GTTs) were performed on rndomly selected suset of nimls (n = 1/group). After injecting 2% (w/v) glucose solution, lood glucose levels were mesured with Contour glucometer (Byer HelthCre LLC, Mishwk, IN, USA) t seline, 15, 3, 6, nd 12 minutes. Also fter the rts were on the diet for 21 weeks, quntittive mgnetic resonnce (qmr) nlysis (EchoMRI, Houston, TX) ws done on rndomly selected suset of nimls (n = 1/group) to otin percent ody ft nd len mss. At 22 weeks on the diet, ll the mice were fsted for 12 hours nd lood smples were collected from the retro-oritl venous plexus. After cogulting t room temperture for 3 minutes, lood smples were centrifuged t 9,3 g for 5 minutes. Serum ws seprted, then snp-frozen nd stored t 8 C until ssyed for hormones. At 23 weeks on the diet, rndomly selected mice (control, n = 7; control + LEU, n = 6;, n = 6; nd + LEU, n = 7) were fsted for 12 hours nd nesthetized y CO 2 inhltion. Blood ws collected y crdic puncture, nd pncret were collected nd stored for nlyses other thn those outlined in this mnuscript. One mouse from the control + LEU group died, nd one mouse from the group died efore week 23. All remining mice (n = 15/group) were sucutneously injected into the right flnk with 5, Pnc2 cells (kindly provided y Dr. J. Schlom, Ntionl Cncer Institute, Bethesd, MD, USA) suspended in serumfree McCoy s 5A medium. Once plple, tumors were mesured weekly with clipers, nd tumor volume ws pproximted using the formul for n ellipsoid (4/3πr 2 1 r 2 ). At 27 weeks on the diet, mice were fsted for 12 hours nd then nesthetized y CO 2 inhltion. They then underwent crdic puncture for lood collection nd were susequently killed y cervicl disloction. Pncretic tumors were hrvested nd either snp-frozen in liquid nitrogen nd stored t 8 C, or fixed with 1% neutrluffered formlin overnight, switched to 7% ethnol,

3 Liu et l. Cncer & Metolism 214, 2:6 Pge 3 of 12 prffin emedded, susequently used for immunohistochemicl nlyses. Serum hormones After study termintion, serum insulin nd leptin levels were nlyzed using Lincoplex ed-sed multiplexed ssys (Millipore, Billeric, MA, USA; MADPK-71 K-7). Serum diponectin nd IGF-1 were quntified y singleplex ssy kits (Millipore; MADPK-71 K-ADPN nd RMIGF187K, respectively). All ssys were nlyzed using BioRd Bioplex nlyzer (BioRd, Hercules, CA, USA) ccording to mnufcturer s directions. Immunohistochemicl nlyses Formlin-fixed tissues were emedded in prffin, cut into 4-μm thick sections, nd processed for immunohistochemistry t the Histology Core Lortory t The U.T. M.D. Anderson Cncer Center, Science Prk Reserch Division (Smithville, TX, USA). Antiodies used for immunohistochemistry were optimized y core personnel using positive nd negtive controls for ech nlysis. Slides were deprffinized in xylene nd sequentilly rehydrted in ethnol to wter. Endogenous peroxidse ctivity ws quenched with 3% hydrogen peroxide for 1 minutes. Antigen retrievl required microwving slides with 1 mm citrte uffer. Nonspecific inding ws locked y treting sections with Biocre locking regent (Biocre Medicl, Concord, CA, USA) for 3 minutes t room temperture, followed y incution with primry ntiody diluted in locking uffer overnight t 4 C. The following primry ntiodies nd dilutions were used: phospho-s6 riosoml protein S235/236 (Cell Signling, Dnvers, MA, USA; 1:1); phospho-mtor Ser2448 (Cell Signling; 1:5); phospho-acc Ser79 (Cell Signling; 1:5); Ki-67 (Dko, Crpinteri, CA, USA; 1:2); cyclin D1 (Snt Cruz Biotechnology, Snt Cruz, CA, USA; 1:5); nd cleved cspse-3 (R&D Systems, Minnepolis, MN, USA; 1:5). Slides were wshed twice in PBS, incuted for 3 minutes with secondry ntiody, wshed two times with PBS, stined with diminoenzidine (BioGenex, Fremont, CA, USA) nd counterstined with hemtoxylin. Imges were cptured y the Aperio ScnScope XT (Aperio Technologies, Vist, CA, USA) nd stining ws quntified using the Aperio ImgeScope (Aperio Technologies). For Ki-67, phospho-mtor, nd cyclin D1 quntifiction, utomted lgorithms were used to determine negtive or positive nucler stining. The percentge of positive cells ws otined with 2 ojective in pncretic tumor sections. Positive stining ws defined s 1+, 2+, nd 3+ for cyclin D1 nd phospho- ACC. Positive stining for phospho-mtor ws defined s only 2+ nd 3+ due to its high seline phosphoryltion. Cleved cspse-3 (CC3) ws quntified s the verge re of positively stined cells, with positive stining defined s t lest 7% of 1 μm 1 μm section. For ll proteins, the positive stining ws verged per group (n = 5/group). In vitro studies Pnc2 cells were cultured in 37 C incutor under 5% CO 2 with McCoy s 5A medi with glutmine (HyClone, Logn, UT, USA) nd 3 g/l glucose ut without BCAA, nd then supplemented with penicillin/streptomycin, nonessentil mino cids, sodium pyruvte, HEPES, 1% hetinctivted FBS (HyClone), nd physiologicl levels of leucine, isoleucine, nd vline (MP Biomedicls, Snt An, CA, USA) [32]. For western lotting, pproximtely 1, Pnc2 cells were seeded in 6-well pltes nd llowed to settle overnight in McCoy s 5A medi with 1% FBS. Cells were then treted with McCoy s 5A plus 1% FBS with or without leucine supplementtion nd McCoy s 5A plus 1% FBS with or without leucine supplementtion (MP Biomedicls, Snt An, CA, USA). For western lot nlysis, cells were treted for 2 min with.3 mm leucine fter 3 hours of medi pretretment. Western lotting Pnc2 cells were lysed on ice for 1 hour in RIPA uffer (Sigm, St. Louis, MO, USA) with protese inhiitor tlet (Roche Applied Sciences, Indinpolis, IN, USA) nd phosphtse inhiitor cocktils II nd III (Sigm). Protein lystes (4 μg) were resolved y SDS-PAGE using 6%, trnsferred to PVDF memrnes (Bio-Rd, Hercules, CA, USA) overnight t 25 volts nd locked for 1 hour t room temperture with LI-COR Blocking Buffer (LI-COR Biotechnologies, Lincoln, NE, USA). Memrnes were incuted overnight t 4 C with primry ntiody (from Cell Signling unless otherwise stted) diluted in 5% BSA (Snt Cruz) nd specific for: phospho-acc Ser79 (1:1), β-ctin (1:1; Sigm), phospho-ampkα T172 (1:1), cleved cspse-3 (1:1), phospho-mtor Ser2448 (1:1), phospho-p7s6k T389 (1:1), nd phospho-s6 riosoml protein Ser235/236 (1:1). β-ctin ws used s loding control for ll ntiodies. After three wshes (5 minutes ech) in.1% Tween-2/PBS (PBS-T), memrnes were incuted for 1 hour t room temperture in species-specific secondry ntiody (LI-COR) diluted (1:5) in LI-COR Blocking Buffer. Following two wshes in PBS-T nd one wsh in PBS, memrnes were scnned using the Odyssey infrred fluorescent imging system (LI-COR). Densitometry ws performed using LI-COR softwre (LI-COR). Reltive levels of proteins were clculted from three iologicl replictes. Cell prolifertion ssy Cell viility ws mesured y the 3-(4,5-dimethyl-2- thizolyl)-2,5-diphenyl-2h-tetrzolium romide (MTT)

4 Liu et l. Cncer & Metolism 214, 2:6 Pge 4 of 12 Cell Prolifertion Assy Kit (Trevigen, Githersurg, MD; K). In 96-well pltes, Pnc2 (15 cells/ well) in medi were llowed to dhere overnight. Ech well ws filled with fresh tretment medi supplemented with different mounts of FBS (1% or 1%) nd leucine ( or.3 mm). The cells were then incuted for 24 h t 37 C, exposed to fresh tretment medi fter the removl of old medi, nd incuted for n dditionl 24 h. MTT ws dded t 1:1 rtio for 2 h, then the liquid ws spirted nd 1 μl of dimethyl sulfoxide (DMSO) ws dded to lyse the cells nd dissolve the solid residue. The opticl density of ech well t 57 nm nd 69 nm, reference wvelength, ws determined using the Synergy 2 Multi-Detection Microplte Reder nd Gen5 Dt Anlysis Softwre (Fisher Scientific, Pittsurgh, PA). Reltive cell viility ws then clculted using the sornce of cells grown in medi with 1% FBS nd no leucine supplementtion for normliztion. Dt shown represent the verge of three iologicl replictes. Sttisticl nlysis Sttisticl nlyses were conducted using GrphPd Prism (GrphPd Softwre, L Joll, CA). Temporl differences etween groups with respect to ody weight nd energy intke were ssessed using repeted mesures nlysis; finl mesurements were compred using one-wy ANOVA nd Newmn-Keul s post hoc test of significnce. Blood glucose levels t ech time point were compred using one-wy ANOVA followed y Newmn-Keul s post hoc test of significnce, nd overll lood glucose differences were compred y performing one-wy ANOVA on clculted res under the curve followed y Newmn-Keul s post hoc test of significnce. Pretumor serum hormone levels, percent ody ft, len mss, nd fsting glucose t week 21 were compred y one-wy ANOVA followed y Newmn-Keul s post hoc test of significnce. Finl mesurements of ex vivo tumor volume nd immunohistochemicl stining of ll ntiodieswerelsocompredmongthegroupsyonewy ANOVA followed y Newmn-Keul s post hoc test of significnce. To compre the effects of leucine supplementtion in medi with either 1% FBS or 1% FBS, western lot densitometry nd reltive cell viilities t their respective time points were compred y two-tiled t-tests. Results were considered significnt if P <.5. Results Effects of clorie restriction nd leucine supplementtion on ody composition, glucose homeostsis, nd serum hormones Mle C57BL/6 mice were fed control diet with or without leucine supplementtion, or 3% diet with or without leucine supplementtion, for 27 weeks (including 21 weeks of diet efore GTTs nd qmrs were performed). Reltive to the control mice, the mice hd significntly reduced cloric intke (n = 22/group; P <.1), ody weight (n = 22/group; P <.1), ody ft (n = 1/group; P <.1), nd len mss (n = 1/group; P <.1), irrespective of leucine supplementtion (Figure 1A-D). At 21 weeks of study, the group without leucine supplementtion, reltive to controls without leucine supplementtion, displyed enhnced glucose clernce s ssessed y GTT (n = 1/group; P <.5), with lood glucose concentrtions peking in 15 minutes in mice nd 3 minutes in control mice following glucose olus (Figure 1E). Leucine supplementtion significntly decresed glucose clernce in the context of the highclorie control diet (n = 1/group; P <.1), ut did not significntly lter glucose uptke in the context of the diet (n = 1/group; P >.5) (Figure 1E). Even t 6 weeks, leucine supplementtion showed the sme trend of inhiiting glucose clernce in mice on the control diet (see Additionl file 1). The diet group without leucine supplementtion showed significntly lower fsting serum glucose levels reltive to the controls (control group, n = 14; group, n = 15; P <.1) (Figure 1F), nd leucine supplementtion further reduced glucose levels in the mice (n = 15/group; P <.1), ut did not ffect glucose levels in control mice (control group, n = 14; control group with leucine supplementtion, n = 15; P >.5). The mice without leucine supplementtion, reltive to the control mice without leucine supplementtion, hd significntly lower serum levels of IGF-1 (control group, n = 9; group, n = 1; P <.1) (Figure 1H) nd leptin (control group, n = 9; group, n = 1; P <.1) (Figure 1I) nd higher levels of diponectin (control group, n = 9; group, n = 1; P <.1) (Figure 1J) ut did not hve significntly ltered levels of insulin (control group, n = 9; group, n = 1; in P >.5) (Figure 1G). Leucine supplementtion lowered IGF-1 in the control group (n = 9/group; P <.1) nd reduced diponectin (n = 1/group; P <.5) in the mice, ut cused no other ltertions to the levels of the other energy-responsive serum hormones mesured in either diet group (Figure 1G-H). Effects of clorie restriction nd/or leucine supplementtion on Pnc2 tumor growth nd poptosis To interrogte whether leucine supplementtion modultes murine pncretic cncer cell growth in control mice, nd/or impcts the nticncer response to, we injected mice from ech diet group with Pnc2 cells t week 23 nd monitored tumor growth during the next 4 weeks. The finl men ex vivo tumor volume from mice, oth with nd without leucine supplementtion, ws significntly smller thn control mice (n = 14/group;

5 Liu et l. Cncer & Metolism 214, 2:6 Pge 5 of 12 A Cloric intke (kcl) C E G I LEU +LEU Time (weeks) Blood glucose (mg/dl) Body ft (%) Serum insulin (ng/ml) Serum leptin (ng/ml) LEU +LEU,c c Time following glucose injection (min) , +LEU,,c +LEU +LEU c +LEU +LEU +LEU B D F H J LEU +LEU Time (weeks) LEU +LEU c,d +LEU +LEU Figure 1 Effects of clorie restriction () nd/or leucine (LEU) supplementtion on ody composition glucose tolernce, nd hormones. (A) Cloric intke nd (B) ody weight of C57BL/6 mle mice on control nd diets with nd without leucine supplementtion reported until glucose tolernce test (GTT) nd quntittive mgnetic resonnce imging (qmri) performed (21 weeks, n = 22/group; P <.1 etween groups with different letters). (C) qmri quntifiction of ody ft nd (D) len mss etween mice fed control or diets with nd without leucine supplementtion for 21 weeks (n = 1/group; P <.1 etween ll groups with different letters). (E) GTT performed fter 21 weeks on diet (n = 1/group; P <.5 etween control nd, P <.1 etween ll other groups with different letters). (F) Fsting glucose levels fter 21 weeks on diet (prior to tumor injection; control, n = 14; ll other groups, n = 15) (P <.1 etween ll groups with different letters). (G-J) Serum hormone nlyses fter 21 weeks on diet (prior to tumor injection) of (G) insulin (P <.5 etween control nd + LEU), (H) IGF-1 (P <.1 etween groups with different letters), (I) leptin (P <.1 etween control nd oth groups; P <.1 etween control + LEU nd oth groups), nd (J) diponectin (P <.1 etween nd oth control groups; P <.5 etween ll other groups with different letters) (control groups, n = 9; groups, n = 1). All dt re presented s the men with error rs indicting the SD (A,B) or SEM (C-J). Differences re considered significnt if P <.5. Arevitions:, control diet;,clorie restriction diet; LEU, leucine-supplemented diet. Body weight (g) Len mss (g) Serum glucose (mg/dl) Serum IGF-1 (ng/ml) Serum diponectin (ug/ml) ,c +LEU c +LEU d +LEU c +LEU

6 Liu et l. Cncer & Metolism 214, 2:6 Pge 6 of 12 P <.1). However, leucine supplementtion resulted in significntly lrger tumors in oth the control nd diet groups, reltive to ech diet s respective nonsupplemented group (control group nd control with leucine supplementtion group, n = 14; P <.1) ( group, n = 14; group with leucine supplementtion, n = 13; P <.1) (Figure 2A). The influence of energy lnce nd leucine supplementtion on cell prolifertion ws ssessed in tumor tissues y immunohistochemicl stining ginst Ki-67 (Figure 2B). While significntly reduced cell prolifertion, reltive to control diet, in nonsupplemented mice (n = 5/group; P <.1), leucine supplementtion significntly incresed cell prolifertion reltive to the respective nonsupplemented mice within oth diet groups (n = 5/ group; P <.1). The mount of Ki-67 stining in the leucine-supplemented group ws ugmented to the level of the nonsupplemented control group (n = 5/group; P <.1). Leucine supplementtion in the group enhnced tumor prolifertion more thn it did tumor urden (Figure 2A,B), suggesting tht finl tumor size ws influenced y oth prolifertion nd poptosis. Bsed on immunohistochemicl nlysis of tumors, we found no pprecile levels of CC3 in tumors from mice not supplemented with leucine; however, leucine supplementtion in oth the control nd diet groups resulted in mrked CC3-positive res (n = 5/group; P <.5) (Figure 2B). Leucine supplementtion in the group resulted in much higher levels of poptosis with 9.8 percent of the tumor composed of poptotic res in the group compred to 1.6 percent in the control group (Figure 2B). This increse in poptosis could explin why leucine supplementtion in the group, despite n equivlent level of Figure 2 Effects of leucine supplementtion on Pnc2 tumor growth nd poptosis. (A) Differences in tumor volume etween mice on control nd clorie restriction () diets with nd without leucine (LEU) supplementtion 4 weeks fter tumor cell injections (control, control + LEU, nd, n = 14/group; nd + LEU, n = 13) (P <.1 etween control nd oth leucine-supplemented groups; P <.1 etween ll other groups with different letters). (B) Comprison of immunohistochemicl nlyses performed on tumor sections for Ki-67 (n = 5/group; P <.1 etween ll groups with different letters) nd cleved-cspse 3 (CC3) (n = 5/group; P <.1 etween the two leucine-supplemented groups; P <.5 etween ll other groups with different letters). Scle rs represent 2 μm. Tumor volume is presented s men ± SD, nd Ki-67 nd CC3 dt re presented s men ± SEM. Differences re considered significnt if P <.5. Arevitions:, control diet;,clorie restriction diet; LEU, leucine-supplemented diet.

7 Liu et l. Cncer & Metolism 214, 2:6 Pge 7 of 12 prolifertion s the control group, resulted in restrined tumor growth. Although poptosis occurred in tumors of mice tht consumed leucine-supplemented diets,.3 mm leucine supplementtion in vitro did not significntly ffect CC3 levels (Additionl file 2) due to 1% FBS only prtilly modeling through growth fctor reduction. nd leucine supplementtion hve differentil effects on energy responsive signling intermedites The effects of energy lnce nd leucine supplementtion on mtor signling were ssessed y immunohistochemicl nlyses of the levels of phospho (p)-mtor, p-acc ( mrker of AMPK ctivity, n upstrem inhiitor of mtor), nd p-s6 nd cyclin D1 (oth downstrem mtor trgets). Bsed on this nlysis, we found tht tumors from mice without leucine supplementtion, reltive to tumors from control mice without leucine supplementtion, displyed incresed levels of p-acc (n = 5/group; P <.5) nd reduced levels of p-mtor (n = 5/group; P <.5) nd its downstrem effector, p-s6 riosoml protein (n = 5/group; P <.1). Additionlly, tumors from mice, reltive to tumors from control mice, showed significntly reduced levels of cyclin D1 (n = 5/group; P <.5) (Figure 3A). Leucine supplementtion in the control diet did not significntly lter mounts of these energy responsive intermedites. However, leucine supplementtion in the diet significntly reduced p-acc (n = 5/group; P <.1) nd incresed p-mtor (n = 5/group; P <.5), p-s6 (n = 5/group; P <.5), nd cyclin D1 (n = 5/group; P <.5) to levels comprle to the nonsupplemented control group (Figure 3A-B). Figure 3 Effects of clorie restriction () nd/or leucine (LEU) supplementtion on energy responsive signls in Pnc2 tumors. (A) Comprison of immunohistochemicl nlyses on tumor sections for phospho-mtor (P <.5 etween groups with different letters), phospho-s6 (P <.1 etween control nd ; P <.1 etween control + LEU nd ; P <.5 etween nd + LEU), cyclin D1 (P <.5 etween groups with different letters), phospho-acc (P <.5 etween the two leucine-supplemented groups; P <.1 etween ll other groups with different letters) (n = 5/group). Scle rs represent 2 μm. All dt re presented s the men ± SEM. Differences re considered significnt if P <.5. Arevitions:, control diet;,clorie restriction diet; LEU, leucine-supplemented diet.

8 Liu et l. Cncer & Metolism 214, 2:6 Pge 8 of 12 Effect of leucine supplementtion on Pnc2 cell lines To confirm the prolifertive effect of leucine supplementtion seen in vivo, in vitro nlyses were performed using the Pnc2 cell line. To model the growth fctor restrictive environment in mice reltive to the overweight control mice s seen in Figure 1H, we grew the cells in medi with either 1% FBS or 1% FBS. Supplementing medi with 1% FBS hs een used to mimic serum growth fctor reduction found in clorie-restricted mice [33]. In the growth fctor-rich environment of medi with 1% FBS, cell viility ws significntly incresed y ~3% with.3 mm leucine supplementtion (P <.5) (Figure 4A). Leucine supplementtion lso incresed cell viility y ~3% in the growth fctor-restricted environment of medi with 1% FBS (P <.1) (Figure 4B). These 3% increses re similr to the increses in Ki-67 seen when compring mice on leucine-supplemented diets to their respective nonsupplemented controls (Figure 2B). This.3 mm concentrtion of leucine were chosen sed on experiments showing tht: i) serum leucine incresed y.3 mm in mice consuming leucinesupplemented diet [34]; nd ii) cell viility of Pnc2 cells significntly incresed with.3 mm leucine supplementtion in vitro (Additionl file 3). Effect of leucine supplementtion on mtor pthwy intermedites In order to understnd the differentil response to leucine supplementtion etween the diet groups with respect to mtor signling, in vitro nlyses were performed using Pnc2 cell lines. Western lot nlyses for the energy responsive intermedites p-ampk, p-acc, p-mtor, p-p7s6k, nd p-s6 reveled tht the effects of leucine supplementtion on cell signling intermedites were impcted y growth fctor vilility. In the growth fctorrich environment of medi with 1% FBS, supplementtion with.3 mm leucine hd no effect on phosphorylted AMPK, ACC, mtor, p7s6k, or S6 riosoml protein (Figure 5A,B). In the 1% FBS setting, leucine supplementtion hd no effect on phosphorylted AMPK or ACC, ut did significntly increse phosphorylted mtor (P <.5) nd its downstrem effector S6 riosoml protein (P <.5). Another downstrem effector of mtor, p-p7s6k, ws lso incresed with leucine supplementtion in the 1% FBS setting, lthough the difference ws not sttisticlly significnt (Figure 5A,C). Discussion Findings in this report demonstrte for the first time tht dietry leucine supplementtion increses growth of pncretic tumors. More specificlly, we show tht leucine supplementtion not only enhnces the protumorigenic nture of high clorie, high crohydrte control diet, ut lso prtilly overcomes the well-estlished nticncer effects of. The mechnisms underlying these leucine-induced protumor effects my e dietdependent, suggested y incresed glucose vilility in overweight mice nd incresed ctivtion of the mtor protein synthetic pthwy in mice. Mice dministered the leucine-supplemented control diet developed the lrgest tumors nd hd the highest level of tumor cell prolifertion of ll four groups. The incresed tumor urden oserved in the leucinesupplemented control group (reltive to controls without leucine supplementtion) occurred without significnt chnges in tumorl poptosis or mtor ctivtion, s evidenced y unchnged levels of oth p-ampk, n upstrem inhiitor of mtor, nd p-s6, downstrem effector of mtor. In overweight control mice, high sl levels of circulting IGF-1 nd tumorl mtor ctivity re consistently found [1]. This high level of ctivity likely lunted ny further increse in mtor ctivtion in response to leucine supplementtion in the control diet, suggesting iologicl threshold ws ttined. This A Reltive cell viility Leucine 1% FBS * - + B Reltive cell viility Leucine ** 1% FBS - + Figure 4 Effects of leucine supplementtion on viility of Pnc2 tumor cells. (A-B) Comprison of reltive viility of cells grown in medi with either (A) 1% fetl ovine serum (FBS) or (B) 1% FBS s ssessed y MTT ssys fter 48 hours of.3 mm leucine supplementtion (* = P <.5, ** = P <.1). All dt re presented s the men ± SEM. Differences re considered significnt if P <.5.

9 Liu et l. Cncer & Metolism 214, 2:6 Pge 9 of 12 Figure 5 Effects of leucine supplementtion on energy responsive signls of Pnc2 tumor cells. (A-C) Western lot nlysis of phosphorylted AMPK, ACC, mtor, p7s6k, nd S6 fter 2 minutes of.3 mm leucine dministrtion fter pretretment with respective medi for 3 hours. Dt shown re representtive lots from three iologicl replictes, nd imges for ech protein re from the sme lot. (B-C) Reltive phosphoryltion of p-ampk, p-acc, p-mtor, p-p7s6k, nd p-s6 in cells grown in medi with either (A) 1% FBS or (B) 1% FBS with or without leucine supplementtion (* = P <.5). All dt re presented s the men ± SEM. Differences re considered significnt if P <.5. concept of iologicl threshold for mtor phosphoryltion ws sustntited using Pnc2 cncer cells in vitro, ecuse mtor ctivtion ws only enhnced in response to leucine under growth fctor restrictive conditions (1% FBS) nd not growth fctor-undnt conditions (1% FBS). The enhnced tumor growth in the leucinesupplemented control group cnnot e explined y chnges in mtor signling in the tumor, ut ws ssocited with greter glucose vilility (reduced fsting insulin levels nd diminished glucose clernce). High levels of glucose hve een shown to increse prolifertion in multiple pncretic cncer cell lines y stimulting glucose consumption nd metolism [35,36]. Although the noted effects on insulin levels in the control group contrdict

10 Liu et l. Cncer & Metolism 214, 2:6 Pge 1 of 12 the puttive chrcteristics of leucine s n insulin secretgogue nd enhncer of lood glucose disposl in ptients with type 2 dietes [37], recent evidence suggests tht leucine s effects on glucose sensitivity differ depending on physiologic context, i.e., dietic versus non-dietic stte [38]. In physiologic scenrio, leucine stimultes mtor ctivity in the β-cells of the pncres nd promotes prolifertion nd thus insulin secretion [38]. On the other hnd, chronic β-cell hyperfunction, consequence of excessive leucine exposure, results in ccelerted β-cell poptosis nd eventul secretory deficiency through negtive feedck loop involving the mtor-dependent inhiition of IRS-1 [39]. Indeed, diet consisting of high levels of leucine comined with sturted ftty cids results in insulin resistnce in rodents [4], nd chronic infusion of mino cids t high concentrtions induces insulin resistnce in humns [41]. Leucine supplementtion did not induce insulin resistnce in mice on the regimen. hs een shown to decrese sl p7s6k ctivtion, which my hve protected ginst mtor-dependent β-cell hyperfunction [4]. Tken together, our dt suggest tht control tumors otined leucine-induced growth dvntge ecuse of incresed glucose vilility s consequence of either impired insulin secretion or function. Mice dministered the diet without leucine supplementtion hd the smllest tumors nd lowest level of tumor cell prolifertion, while mice fed the leucinesupplemented regimen (reltive to mice without leucine supplementtion) hd incresed tumor growth to levels intermedite etween the unsupplemented mice on the nd control diets. Leucine supplementtion in the diet, reltive to lone, lso incresed tumor cell prolifertion (to the levels oserved in control mice), nd incresed poptosis. It is not uncommon to oserve increses in oth cell prolifertion nd cell deth in the sme tumor, s seen in the tumors of mice on the diet. In fct, numer of dominnt oncogenes tht increse prolifertion through induction of errnt growth signls, lso induce poptosis [42]. Thus, leucineinduced dysregultion of growth signls, such s mtor ctivtion, in setting of low-energy sustrtes nd growth fctors in response to regimen, my explin the oserved increses in poptosis, tumor cell prolifertion (to control levels), nd prtil rescue of tumor urden in the leucine-supplemented mice. This rescue of tumor urden ws only prtil, ecuse leucine significntly incresed prolifertion. As previously stted, high levels of mtor ctivity support prolifertion nd survivl of pncretic cncer cells, nd consistently results in decresed ctivtion of mtor in pncretic tumors [13]. In contrst to tumors from the leucine-supplemented control group, we found tht tumors from the leucinesupplemented group demonstrted mrked increses in mtor ctivtion, s evidenced y lower levels of p- AMPK nd higher levels of p-s6 nd cyclin D1, without chnges in fsting insulin levels nd glucose clernce. The mintennce of physiologic insulin secretion in the mice ws perhps due to the protection of β-cells y chronic, strtegy tht hs een shown to increse β-cell prolifertion in rts [43]. Tken together, our dt suggest tht tumors otined leucineinduced growth dvntge ecuse of incresed mtor ctivtion. Conclusions This report estlishes tht dietry leucine supplementtion, irrespective of energy lnce sttus, promotes pncretic tumor growth. These findings suggest cution regrding the clinicl use of leucine supplementtion for purposes of len muscle enhncement in cchectic cncer ptients. Additionl reserch is needed to scertin the impct mino cids (BC or otherwise) hve on cncer growth nd muscle repir; the identifiction of mtorindependent pproches to spre muscle in cchectic cncer ptients; nd the link etween energy lnce, mtor signling, nd mino cid metolism. Additionl files Additionl file 1: Effects of leucine supplementtion on glucose tolernce t 6 weeks. Glucose tolernce test (GTT) performed fter 6 weeks on diet (n = 1/group; P <.1 etween control with leucine supplementtion nd the clorie restriction () groups; P <.5 etween the control groups). All dt re presented s the men ± SEM. Differences re considered significnt if P <.5. Additionl file 2: Effects of single BCAA supplementtion on poptosis of Pnc2 tumor cells. Western lot nlysis of cleved cspse-3 protein levels fter 24 hours of either.3 mm leucine, isoleucine, or vline dministrtion. Dt shown re representtive lots from three iologicl replictes. Reltive protein levels of cleved cspse-3 were quntified y densitometry using LI-COR Odyssey softwre. All dt re presented s the men ± SEM. Differences re considered significnt if P <.5. Additionl file 3: Effects of different doses of leucine supplementtion on Pnc2 tumor cell viility. Comprison of reltive cell viility s ssessed y MTT ssys fter 48 hours of leucine supplementtion (* = P <.5). All dt re presented s the men ± SEM. Differences re considered significnt if P <.5. Arevitions ACC: cetyl-coa croxylse; AIN: Americn Institute of Nutrition; AMPK: AMP-ctivted protein kinse; BCAA: rnched-chin mino cid; BSA: ovine serum lumin; CC3: cleved cspse-3; : clorie restriction; FBS: fetl ovine serum; GTT: glucose tolernce test; IGF-1: insulin-like growth fctor-1; IRS-1: insulin receptor sustrte-1; mtor: mmmlin trget of rpmycin; p7s6k: p7s6 kinse; PBS: phosphte-uffered sline; PBS-T: phosphte-uffered sline with tween; PI: propidium iodide; PVDF: polyvinylidene difluoride; qmri: quntittive mgnetic resonnce imging; RIPA: rdioimmunoprecipittion ssy; SDS-PAGE: sodium dodecyl sulphte-polycrylmide gel electrophoresis. Competing interests The uthors declre tht they hve no conflicts or competing interests.

11 Liu et l. Cncer & Metolism 214, 2:6 Pge 11 of 12 Authors contriutions KAL conducted in vitro nlysis, including western lot nlysis nd DNA leling, nlyzed IHC stining of tumor tissue, performed some of the sttisticl nlysis, nd drfted the mnuscript. LML prticipted in study design, performed some of the niml techniques, nlyzed portion of the IHC dt, nd edited the mnuscript. AJR designed nd conducted the niml study, performed some of the sttisticl nlysis, nd edited the mnuscript. SDH designed the study nd edited the mnuscript. All uthors red nd pproved the finl mnuscript. Acknowledgements This study ws supported y n NIH grnt (R1 CA wrded to SDH), the Winkler Fmily Foundtion, nd NIH-sponsored postdoctorl fellowship grnts (R25T CA5773 nd T32 CA wrded to LML). Funding from R1 supported AJR nd KAL. The funding ody plyed no role in the design, collection, nlysis, interprettion of dt, writing, or decision to pulish. We thnk Dr. Schlom for providing Pnc2 murine pncretic cell lines, the histology core t U.T. M.D.A. Cncer Center (Smithville, TX) for performing IHC stining, nd Michelle Rmsey for dministrtive support. Received: 3 Octoer 213 Accepted: 18 Mrch 214 Pulished: 31 Mrch 214 References 1. Jeml A, Siegel R, Wrd E, Ho Y, Xu J, Thun MJ: Cncer sttistics. CA Cncer J Clin 29, 59: Lillemoe KD, Yeo CJ, Cmeron JL: Pncretic cncer: stte-of-the-rt cre. CA Cncer J Clin 2, 5: Clle EE, Rodriguez C, Wlker-Thurmond K, Thun MJ: Overweight, oesity, nd mortlity from cncer in prospectively studied cohort of U.S. dults. N Engl J Med 23, 348: Gpstur SM, Gnn PH, Lowe W, Liu K, Colngelo L, Dyer A: Anorml glucose metolism nd pncretic cncer mortlity. JAMA 2, 283: Silvermn DT, Swnson CA, Gridley G, Wcholder S, Greenerg RS, Brown LM, Hyes RB, Swnson GM, Schoenerg JB, Pottern LM, Schwrtz AG, Frumeni JF Jr, Hoover RN: Dietry nd nutritionl fctors nd pncretic cncer: cse control study sed on direct interviews. J Ntl Cncer Inst 1998, 9: Bjornsti MA, Houghton PJ: The TOR pthwy: trget for cncer therpy. Nt Rev Cncer 24, 4: Hrrison DE, Strong R, Shrp ZD, Nelson JF, Astle CM, Flurkey K, Ndon NL, Wilkinson JE, Frenkel K, Crter CS, Phor M, Jvors MA, Fernndez E, Miller RA: Rpmycin fed lte in life extends lifespn in geneticlly heterogeneous mice. Nture 29, 46: Hursting SD, Smith SM, Lshinger LM, Hrvey AE, Perkins SN: Clories nd crcinogenesis: lessons lerned from 3 yers of clorie restriction reserch. Crcinogenesis 21, 31: Kphi P, Zid BM, Hrper T, Koslover D, Spin V, Benzer S: Regultion of lifespn in drosophil y modultion of genes in the TOR signling pthwy. Curr Biol 24, 14: Lshinger LM, Mlone LM, McArthur MJ, Golderg JA, Dniels EA, Pvone A, Coly JK, Smith NC, Perkins SN, Fischer SM, Hursting SD: Genetic reduction of insulin-like growth fctor-1 mimics the nticncer effects of clorie restriction on cyclooxygense-2-driven pncretic neoplsi. Cncer Prev Res 211, 4: Velli T, Tkcs-Velli K, Zhng Y, Kovcs AL, Orosz L, Muller F: Genetics: influence of TOR kinse on lifespn in C. elegns. Nt 23, 426: Petroulkis E, Mmne Y, Le Bcquer O, Shhzin D, Sonenerg N: mtor signling: implictions for cncer nd nticncer therpy. Br J Cncer 26, 94: Lshinger LM, Mlone LM, Brown GW, Dniels EA, Golderg JA, Otto G, Fischer SM, Hursting SD: Rpmycin prtilly mimics the nticncer effects of clorie restriction in murine model of pncretic cncer. Cncer Prev Res 211, 4: Bjotto G, Sto Y, Kitur Y, Shimomur Y: Effect of rnched-chin mino cid supplementtion during unloding on regultory components of protein synthesis in trophied soleus muscles. Eur J Appl Physiol , 211: Blomstrnd E, Elisson J, Krlsson HK, Kohnke R: Brnched-chin mino cids ctivte key enzymes in protein synthesis fter physicl exercise. J Nutr 26, 136:269S 273S. 16. Li F, Yin Y, Tn B, Kong X, Wu G: Leucine nutrition in nimls nd humns: mtor signling nd eyond. Amino Acids 211, 41: Proud CG: mtor-medited regultion of trnsltion fctors y mino cids. Biochem Biophys Res Commun 24, 313: Sns MD, Tshiro M, Vogel NL, Kimll SR, D Alecy LG, Willims JA: Leucine ctivtes pncretic trnsltionl mchinery in rts nd mice through mtor independently of CCK nd insulin. J Nutr 26, 136: Churchwrd-Venne TA, Burd NA, Phillips SM: Nutritionl regultion of muscle protein synthesis with resistnce exercise: strtegies to enhnce nolism. Nutr Met 212, 9:4. 2. Tisdle MJ: Mechnisms of cncer cchexi. Physiol Rev 29, 89: Feron KC, Brcos VE: Cchexi in pncretic cncer: new tretment options nd mesures of success. HPB (Oxford) 21, 12: Deutz NE, Sfr A, Schutzler S, MemelinkR,FerrndoA,SpencerH,vnHelvoort A, Wolfe RR: Muscle protein synthesis in cncer ptients cn e stimulted with specilly formulted medicl food. Clin Nutr 211, 3: Feron K, Arends J, Brcos V: Understnding the mechnisms nd tretment options in cncer cchexi. Nt Rev Clin Oncol 213, 1: McNurln MA, Heys SD, Prk KG, Broom J, Brown DS, Eremin O, Grlick PJ: Tumour nd host tissue responses to rnched-chin mino cid supplementtion of ptients with cncer. Clin Sci 1994, 86: Peters SJ, vn Helvoort A, Kegler D, Argiles JM, Luiking YC, Lvino A, vn Bergenhenegouwen J, Deutz NE, Hgsmn HP, Gorselink M, vn Norren K: Dose-dependent effects of leucine supplementtion on preservtion of muscle mss in cncer cchectic mice. Oncol Rep 211, 26: Nishio Y, Kkizoe T, Ohtni M, Sto S, Sugimur T, Fukushim S: L-isoleucine nd L-leucine: tumor promoters of ldder cncer in rts. Science 1986, 231: Xie XL, Wei M, Yunoki T, Kkehshi A, Ymno S, Kto M, Wniuchi H: Long-term tretment with L-isoleucine or L-leucine in AIN-93G diet hs promoting effects on rt ldder crcinogenesis. Food Chem Toxicol 212, 5: Nunez NP, Perkins SN, Smith NC, Berrign D, Berendes DM, Vrticovski L, Brrett JC, Hursting SD: Oesity ccelertes mouse mmmry tumor growth in the sence of ovrin hormones. Nutr Cncer 28, 6: Whetley KE, Nogueir LM, Perkins SN, Hursting SD: Differentil effects of clorie restriction nd exercise on the dipose trnscriptome in diet-induced oese mice. J Oes 211, 211: Guillet C, Zngrelli A, Mishellny A, Rousset P, Sornet C, Drdevet D, Boirie Y: Mitochondril nd srcoplsmic proteins, ut not myosin hevy chin, re sensitive to leucine supplementtion in old rt skeletl muscle. Exp Gerontol 24, 39: Ventrucci G, Mello MA, Gomes-Mrcondes MC: Protesome ctivity is ltered in skeletl muscle tissue of tumour-ering rts leucine-rich diet. Endocr Relt Cncer 24, 11: Cccitore L, De Mrco F, Cerini R, De Ritis F: Free mino cids in plsm during experimentl infection of mice with the MHV-3 strin of mouse heptitis virus. J Infect Dis 1977, 136: Lee C, Rffghello L, Brndhorst S, Sfdie FM, Binchi G, Mrtin-Montlvo A, Pistoi V, Wei M, Hwng S, Merlino A, Emionite L, de Co R, Longo VD: Fsting cycles retrd growth of tumors nd sensitize rnge of cncer cell types to chemotherpy. Sci Trnsl Med 212, 4:124r Mcotel Y, Emnuelli B, Bng AM, Espinoz DO, Boucher J, Beee K, Gll W, Khn : Dietry leucine n environmentl modifier of insulin resistnce cting on multiple levels of metolism. PLoS One 211, 6:e Hn L, M Q, Li J, Liu H, Li W, M G, Xu Q, Zhou S, Wu E: High glucose promotes pncretic cncer cell prolifertion vi the induction of EGF expression nd trnsctivtion of EGFR. PLoS One 211, 6:e Liu H, M Q, Li J: High glucose promotes cell prolifertion nd enhnces GDNF nd RET expression in pncretic cncer cells. Mol Cell Biochem 211, 347: Leenders M, vn Loon LJ: Leucine s phrmconutrient to prevent nd tret srcopeni nd type 2 dietes. Nutr Rev 211, 69: Zoncu R, Efeyn A, Stini DM: mtor: from growth signl integrtion to cncer, dietes nd geing. Nt Rev Mol Cell Biol 211, 12: Melnik BC: Leucine signling in the pthogenesis of type 2 dietes nd oesity. World J Dietes 212, 3:38 53.

12 Liu et l. Cncer & Metolism 214, 2:6 Pge 12 of Znchi NE, Guimres-Ferreir L, Siqueir-Filho MA, Griel Cmporez JP, Nicstro H, Seixs Chves DF, Cmpos-Ferrz P, Lnch AH Jr, de Oliveir Crvlho : The possile role of leucine in modulting glucose homeostsis under distinct ctolic conditions. Med Hypotheses 212, 79: Kres M, Brunmir B, Brehm A, Artwohl M, Szendroedi J, Nowotny P, Roth E, Furnsinn C, Promintzer M, Anderwld C, Bischof M, Roden M: The mmmlin trget of rpmycin pthwy regultes nutrient-sensitive glucose uptke in mn. Dietes 27, 56: Alenzi FQ: Links etween poptosis, prolifertion nd the cell cycle. Br J Biomed Sci 24, 61: He XY, Zho XL, Gu Q, Shen JP, Hu Y, Hu RM: Clorie restriction from young ge preserves the functions of pncretic et cells in ging rts. Tohoku J Exp Med 212, 227: doi:1.1186/ Cite this rticle s: Liu et l.: Leucine supplementtion differentilly enhnces pncretic cncer growth in len nd overweight mice. Cncer & Metolism 214 2:6. 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