Applied Physiology, Nutrition, and Metabolism

Transcription

1 Applied Physiology, Nutrition, nd Metolism Evluting the Effect of 20-Hydroxyecdysone (20HE) on Mechnistic Trget of Rpmycin Complex 1 (mtorc1) Signling in the Skeletl Muscle nd Liver of Rts Journl: Applied Physiology, Nutrition, nd Metolism Mnuscript ID pnm r2 Mnuscript Type: Brief communiction Dte Sumitted y the Author: 12-Sep-2015 Complete List of Authors: Anthony, Trcy; Rutgers University, Nutritionl Sciences Mirek, Emily; Rutgers University, Nutritionl Sciences Brgoud, Alert; Rutgers University, Nutritionl Sciences Phillipson-Weiner, Lindsey; Rutgers University, Nutritionl Sciences DeOliveir, Christopher; Rutgers University, Nutritionl Sciences Wetstein, Berish; Rutgers University, Nutritionl Sciences Grf, Brittny; Rutgers University, Plnt Biology nd Pthology Kuhn, Peter; Rutgers University, Plnt Biology nd Pthology Rskin, Ily; Rutgers University, Plnt Biology nd Pthology Keyword: leucine, mtor, phytoecdysteroids, ergogenic ids < nutrition, sports nutrition < nutrition

2 Pge 1 of 16 Applied Physiology, Nutrition, nd Metolism 1 2 Evluting the Effect of 20-Hydroxyecdysone (20HE) on Mechnistic Trget of Rpmycin Complex 1 (mtorc1) Signling in the Skeletl Muscle nd Liver of Rts Anthony, Trcy G. 1, Mirek, Emily T. 1, Brgoud, Alert Rouf 1, Phillipson-Weiner, Lindsey 1, DeOliveir, Christopher M. 1, Wetstein, Berish 1, Grf, Brittny L. 2, Kuhn, Peter E. 2, Rskin, Ily Deprtment of Nutritionl Sciences nd 2 Deprtment of Plnt Biology nd Pthology, Rutgers University, New Brunswick, NJ Corresponding Author: 10 Trcy G. Anthony, PhD 11 Associte Professor 12 Deprtment of Nutritionl Sciences 13 Forn Hll Room Dudley Rd. 15 New Brunswick, NJ trcy.nthony@rutgers.edu 18 1

3 Applied Physiology, Nutrition, nd Metolism Pge 2 of ABSTRACT Phytoecdysteroids such s 20HE re nutritionl supplements mrketed s enhncers of len ody mss. In this study the impct of 20HE ingestion on protein kinse B/Akt-mTORC1 signling in the skeletl muscle nd liver of mle rts ws found to e limited. Biovilility of 20HE, whether consumed lone or with leucine, lso remined low t ll doses ingested. Additionl work is necessry to clrify 20HE mechnism of ction in vivo KEYWORDS Phytoecdysteroids, PKB/Akt, mtor, 4E-BP1, leucine 2

4 Pge 3 of 16 Applied Physiology, Nutrition, nd Metolism 29 INTRODUCTION Phytoecdysteroids re clss of polyhydroxylted ketosteroid compounds found in plnts nd insects. In rthropods, they function s ndrogens nd re involved in reproduction nd the molting process (Dinn 2009). In plnts, ecdysteroids serve s defense mechnism in which their ingestion cn disrupt n invding insect s hormone lnce, resulting in lethl metolic dmge to the invder (Dinn 2009). One of the most commonly studied ecdysteroids, 20-Hydroxyecdysone (20HE), is nturlly present in foods such s spinch nd quino nd is mrketed s n nolic gent in vrious nutritionl supplements tken to improve thletic performnce (Lfont nd Dinn 2003). Ingestion of 20HE reportedly increses the size of muscle fiers in rts (Toth et l. 2008) s well s incresed len ody mss in sheep, pigs nd quil (Chermnykh et l. 1988; Lfont nd Dinn 2003) nd is ssocited with nti- 39 dietic effects in oese mice (Kizelsztein et l. 2009). Reports in the literture clim nolic effects in nimls nd humns without ndrogenicity (Chermnykh et l. 1988; Lfont nd Dinn 2003; Gorelick- Feldmn et l. 2008), ut one humn tril ws unle to sustntite the nolic potentil of 20HE (Wilorn et l. 2006). The moleculr pthwy mediting 20HE s nolic potentil remins unknown. Despite eing clssified s steroid, 20HE is not structurlly close enough to ndrogens to e le to ind to intrcellulr steroid receptors in humns (Bthori et l. 2008). Insted, it is suggested tht 20HE inds to G protein coupled receptors (GPCR) on the plsm memrne (Bthori et l. 2008). An in vitro study showed tht ecdysteroids increse protein synthesis in muscle cells through n influx of C 2+ nd susequent ctivtion of protein kinse B/Akt (Gorelick-Feldmn et l. 2010). In this study, the effects of 20HE were suppressed y inhiitors of GPCR, phospholipse C (PLC) nd phosphoinositide kinse-3 (PI3K). Seprte from its effects in muscle, other work hs descried nti-oesity nd nti-dietic effects of 20HE in mice vi reduced heptic glucose production in ssocition with incresed Akt phosphoryltion (Kizelsztein et l. 2009). It is unknown if reported chnges in heptic glucose 3

5 Applied Physiology, Nutrition, nd Metolism Pge 4 of production y 20HE re linked in ny wy to cute ctivtion of Akt. Furthermore, it is unknown if 20HE ctivtes downstrem nolic signling seprte from metolic signling in liver. There re no studies ssessing phosphoryltion of Akt y 20HE in vivo. Stimultion of muscle protein synthesis y insulin nd/or insulin-like growth fctor I (IGF-I) is medited vi PI3K-Akt ctivtion of mmmlin trget of rpmycin complex 1 (mtorc1) ssemly nd signl trnsduction (Lplnte nd Stini 2012). Orl ingestion of mino cids, nd in prticulr leucine, lso stimultes mtorc1 ssemly nd signling ut in PI3K-Akt-independent mechnism, llowing for convergence of mino cid nd insulin/igf-i signling on mtorc1 upon mel feeding (Anthony et l. 2000; Anthony et l. 2001; Anthony et l. 2001). Downstrem effectors of mtorc1 such s eukryotic initition fctor 4E inding protein 1 (4E-BP1) function to increse the efficiency of mrna trnsltion initition. To wht extent 20HE lone or in comintion with leucine ctivtes mtorc1 signling in skeletl muscle nd liver is unknown. The ojective of this study ws to ssess the ility of 20HE to stimulte mtorc1 signling in skeletl muscle nd liver. A more thorough understnding of 20HE s moleculr pthwy of ction in vivo cn inform future interventions in humns or nimls MATERIALS AND METHODS Animls nd Study Design. All rts were cred for in ccordnce with the Guide to the Cre nd Use of Lortory Animls nd ll experiments were reviewed nd pproved y the Institutionl Animl Cre nd Use Committee t Rutgers University. Overnight fsted 4-8 week old mle Sprgue-Dwley rts (n=5-6 per group) were rndomized to one of three study designs: 1) gvged with 0, 10, 50, 200 mg/kg 20HE nd euthnized 30 min post-gvge; 2) gvged with 200 mg/kg 20HE or excipient nd euthnized t pre-gvge or 30 min, 60 min, 120 min, 240 min post-gvge, nd 3) dministered excipient or 200 mg/kg 20HE lone or in comintion with 1.35 g/kg L-leucine nd euthnized 30 min post-gvge. In these studies, 3% DMSO 4

6 Pge 5 of 16 Applied Physiology, Nutrition, nd Metolism ws used s excipient in Study 1 nd 2 wheres the emulsifier Lrsol (Hu et l. 2001) ws the excipient in Study 3. For ll studies the volume dministered ws 1 ml/100 g ody weight. Before food removl, rts were mintined in temperture-controlled (23-25 C) room with 12:12h light:drk cycle nd freely-provided commercil rodent diet Smple collection. Rts were euthnized y decpittion nd trunk lood ws collected for nlysis of serum leucine nd 20HE concentrtions. The liver nd hind-lim muscles (gstrocnemius + plntris) were quickly extrcted, weighed nd frozen in liquid nitrogen efore storge in -80⁰C freezer Serum mesurements. Serum leucine ws nlyzed vi HPLC using ortho-phthlldehyde/9- fluorenylmethyl chloroformte (OPA/FMOC) derivtized mino cid nlysis. Detection ws performed ccording to Agilent protocol pp note: EN using Agilent ZORBAX Eclipse Plus C18 column. To quntify iovilility of 20HE, 500 µl liquots of serum smples were extrcted with 500 µl of utnol three times. The orgnic lyer ws dried nd dissolved in 100 µl of 70% ethnol; 5 µl ws injected in LC-MS (5-95% cetonitrile grdient). A stndrd curve of 20HE (5, 10, 50 ng) ws run longside smples in LC-MS for quntifiction Immunolot Anlysis. Tissue smples were prepred for SDS-PAGE followed y immunolot nlysis s previously descried (Anthony et l. 2007). Immunolots included tissue smple from rts injected with IGF-I s positive control for Akt phosphoryltion. Memrnes were incuted with the following primry ntiodies: Akt (p-thr308), mtorc1 (p-ser2448), nd 4E-BP1. Protein expression ws visulized using enhnced chemiluminescence (ECL) nd signl intensities were digitlly cptured using FluorChem M multiplex imger (ProteinSimple) nd nd densities were quntitted using imging softwre. Sttistics. Dt re expressed s mens ± SEM. Experimentl results were nlyzed y ANOVA using the STATISTICA softwre pckge. Differences etween group mens were ssessed y Tukey s post hoc test. The level of significnce ws set t P 0.05 for ll sttisticl tests. 5

7 Applied Physiology, Nutrition, nd Metolism Pge 6 of RESULTS Study 1: 20HE Dose Response Young mle rts were overnight (12h) fsted then gvged with doses of 20HE tht hve een cited in the literture s imprting nolic or metoliclly-fvorle properties in rodents. The lowest dose dministered ws twice the dosge used in previous study reporting nolic properties, nd the highest dose dministered ws 200 mg/kg, n mount higher thn commonly suggested dose in humns of 200 mg per dy, ut sustntilly elow the orl LD50 in mice of >9,000 mg/kg. None of the doses dministered significntly ltered the phosphoryltion sttes of Akt, mtor or 4E-BP1 t 30 minutes fter ingestion in either the skeletl muscle (Figure 1, A-C) or liver (Figure 2, A-C). Quntifiction of 20HE from plsm smples showed there were miniml differences in iovilility mong the doses t 30 minutes when delivered in 3% DMSO in sline. Serum 20HE concentrtions were detectile only in rts 115 gvged with 20HE ut remined similrly low cross ll doses (~0.45 ng/µl) Study 2: 20HE Time Course To determine if the effect of 20HE on Akt phosphoryltion or mtorc1 signling in skeletl muscle nd/or liver ws delyed pst 30 minutes, rts were overnight fsted nd then dministered 200 mg/kg 20HE nd euthnized t 30 min, 60 min, 120 min nd 240 min following gvge. No chnges in the phosphoryltion sttus of Akt, mtor or 4E-BP1 s compred to untreted controls were noted in either skeletl muscle (Figure 1, D-F) or liver (Figure 2, D-F) over the 4 h time course except phosphoryltion of 4E-BP1 ws reduced t 240 min in skeletl muscle only (Figure 1F). Quntifiction of 20HE from plsm showed 20HE levels in lood rnged etween ng/µl fter gvge when delivered in 3% DMSO in sline Study 3: Acute Effects of 20HE versus Leucine 6

8 Pge 7 of 16 Applied Physiology, Nutrition, nd Metolism Previous work y this l nd others show tht orl dministrtion of 1.35 g/kg ody weight L-leucine ctivtes mtorc1 signling in skeletl muscle etween minutes following ingestion without ltering Akt phosphoryltion (Anthony et l. 2001). To compre the impct of 20HE ingestion to tht of leucine, mice were orlly dministered either 20HE (200 mg/kg), leucine (1.35 g/kg), the comintion together, or excipient lone. To improve iovilility of 20HE, Lrsol ws used s the gvge excipient insted of 3% DMSO in sline. Averge serum concentrtions of leucine were sttisticlly similr to verge serum concentrtions of leucine in rts gvged with 20HE. Serum leucine concentrtions rose significntly in rts gvged with leucine lone nd in mice gvged with leucine plus 20HE (oth P<0.05 s compred to control). While leucine concentrtions rose significntly regrdless of 20HE consumption, 20HE iovilility ppered to e improved y Lrsol, though with gret vrition, for serum levels of 20HE mesured 0.74 ± ng/µl in rts gvged with 20HE + leucine compred with 1.52 ± 1.23 ng/µl in rts gvged with 20HE lone. In greement with previous reports, leucine lone significntly incresed phosphoryltion of mtor nd 4E-BP1 in oth skeletl muscle (Figure 1, H-I) nd liver (Figure 2, H-I). On the other hnd, ingestion of 20HE lone did not lter mtorc1 signling in skeletl muscle or liver nd slightly dmpened stimultion of mtorc1 y leucine in skeletl muscle. Neither leucine nor 20HE ltered phosphoryltion of Akt in liver or muscle t 30 min when gvged lone or in comintion (Figure 1G, 2G) DISCUSSION Phytoecdysteroids re one of the most undnt steroid clsses in nture, re non-toxic to mmmls, yet disply pleiotropic effects which re potentilly eneficil if hrnessed properly. Among the postulted helth enefits of this clss of compounds, incresed musculr mss nd improved glucose homeostsis is evidenced in oth in vitro nd in vivo studies (Gorelick-Feldmn et l. 2008; Toth et l. 2008; Kizelsztein et l. 2009; Gorelick-Feldmn et l. 2010; Seidlov-Wuttke et l. 2010). To understnd if mtorc1 signling my contriute to ny of these previously reported outcomes, we emrked on series of experiments evluting cute effects of 20HE consumption in the skeletl muscle nd liver of 7

9 Applied Physiology, Nutrition, nd Metolism Pge 8 of mle rts. Bsed on recent reports descriing rpid ctivtion of Akt ssocited with incresed protein synthesis in cultured myocytes (Gorelick-Feldmn et l. 2010) nd reduced heptic glucose production nd ody weight in oese mice (Kizelsztein et l. 2009), we hypothesized tht ctivtion of Akt y 20HE leds to incresed signling vi the mtorc1 pthwy in skeletl muscle nd/or liver. Yet, study results did not support this ide, showing tht 20HE does not cutely ctivte Akt phosphoryltion or mtorc1 signling in hind lim muscle or liver. As such, the mechnism to explin potentil helth enefits of 20HE remins n open question requiring dditionl explortion. The following nrrtive offers lternte pproches nd dditionl considertions. The iovilility of 20HE ws quite low in these studies nd ppered insensitive to incresing doses. While using n excipient such s Lrsol improved iovilility, this did not result in improved ctivtion of the Akt-mTORC1 pthwy t the time points exmined. The choice to focus on erly time points ws sed on previous work showing ctivtion of Akt y 20HE within 4 h nd ctivtion of mtorc1 y leucine within 1 h (Anthony et l. 2001; Gorelick-Feldmn et l. 2010). A follow-up time course dministering 20HE in comintion with clories nd/or crohydrtes is wrrnted to test the ide tht 20HE functions to ugment insulin/igf-i signling y mel feeding. Interestingly, in the current study provision of leucine in comintion with 20HE results in dmpening of mtorc1 signling despite elevted circulting leucine. Thus, if 20HE is to ugment the ction of nother nutrient, it is not likely to e n mino cid-driven pthwy. Follow up studies exploring how the food mtrix impcts 20HE sorption nd potentil ugmenttion of mcronutrient metolism is wrrnted. This study contriutes importntly to the study of phytoecdysteroids nd estlishes tht cute iovilility of 20HE is limited when consumed lone or with leucine. While one interprettion is tht the nolic signling effect of 20E is limited y digestiility nd sorption, n lternte explntion is tht the mechnism of effect is independent of mtor. Certinly, mong the pthwys ffected y nutritionl sttus, mtorc1 is one of the most importnt cellulr signling hus in the ody, regulting cell growth, utophgy, nd prolifertion (Lplnte nd Stini 2012). However, the pth to improved ody composition does not hve to e medited through mtor. Alternte mechnisms requiring future 8

10 Pge 9 of 16 Applied Physiology, Nutrition, nd Metolism investigtion include phospholipse C nd camp/pka (Gorelick-Feldmn et l. 2010), reducing protein rekdown (e.g., similr to the leucine metolite β-hydroxy β-methylutyrte (Molfino et l. 2013)), ltering vitmin D sttus or ction in muscle (Toth et l. 2010) nd incresing lipolysis so s to improve len ody mss. In summry, study results show tht ingestion of 20HE does not cutely ctivte mtorc1 signling in muscle or liver. Additionl work is necessry to clrify the mechnism y which phytoecdysteroids cn fcilitte muscle growth nd liver metolism in mmmls Conflict of Interest Disclimer: The uthors declre tht there re no conflicts of interest ACKNOWLEDGEMENTS The uthors grtefully cknowledge the expert technicl ssistnce of Pengxing She, PhD, Griel Wilson, PhD, Rn Al Bghddi nd George Wei. This work ws funded y USDA Multistte Project 192 NC1184 nd the New Jersey Agriculturl Experiment Sttion t Rutgers University

11 Applied Physiology, Nutrition, nd Metolism Pge 10 of REFERNCES Anthony, J., Anthony, T., Kimll, S., nd Jefferson, L Signling pthwys involved in trnsltionl control of protein synthesis in skeletl muscle y leucine. J. Nutr. 131: 856S-860S. PMID: Anthony, J., Yoshizw, F., Anthony, T., Vry, T., Jefferson, L., nd Kimll, S Leucine stimultes trnsltion initition in skeletl muscle of postsorptive rts vi rpmycin- sensitive pthwy. J. Nutr. 130(10): PMID: Anthony, T., Anthony, J., Yoshizw, F., Kimll, S., nd Jefferson, L Orl dministrtion of leucine stimultes riosoml protein mrna trnsltion ut not glol rtes of 206 protein synthesis in the liver of rts. J. Nutr. 131(4): PMID: Anthony, T.G., McDniel, B.J., Knoll, P., Bunpo, P., Pul, G.L., nd McNurln, M.A Feeding mels contining soy or whey protein fter exercise stimultes protein synthesis nd trnsltion initition in the skeletl muscle of mle rts. J. Nutr. 137(2): PMID: Bthori, M., Toth, N., Hunydi, A., Mrki, A., nd Zdor, E Phytoecdysteroids nd nolic-ndrogenic steroids--structure nd effects on humns. Curr. Medicinl Chem. 15(1): doi: / #sthsh.Ceoi3oxK.dpuf. PMID:

12 Pge 11 of 16 Applied Physiology, Nutrition, nd Metolism Chermnykh, N.S., Shimnovskii, N.L., Shutko, G.V., nd Syrov, V.N [The ction of methndrostenolone nd ecdysterone on the physicl endurnce of nimls nd on protein metolism in the skeletl muscles]. Frmkol. Toksikol. 51(6): PMID: Dinn, L The Krlson Lecture. Phytoecdysteroids: wht use re they? Arch. Insect Biochem. Physiol. 72(3): doi: /rch PMID: Gorelick-Feldmn, J., Cohick, W., nd Rskin, I Ecdysteroids elicit rpid C2+ flux leding to Akt ctivtion nd incresed protein synthesis in skeletl muscle cells. Steroids (10): doi: /j.steroids PMID: Gorelick-Feldmn, J., Mclen, D., Ilic, N., Poulev, A., Lil, M.A., Cheng, D., nd Rskin, I Phytoecdysteroids increse protein synthesis in skeletl muscle cells. J. Agric. Food. Chem. 56(10): doi: /jf073059z. PMID: Hu, Z., Tw, R., Konishi, T., Shit, N., nd Tkd, K A novel emulsifier, lrsol, enhnces gstrointestinl sorption of gentmicin. Life Sci. 69(24): PMID: Kizelsztein, P., Govorko, D., Komrnytsky, S., Evns, A., Wng, Z., Ceflu, W.T., nd Rskin, I Hydroxyecdysone decreses weight nd hyperglycemi in diet-induced oesity mice model. Am. J. Physiol. Endocrinol. Met. 296(3): E433-E439. doi: /jpendo PMID:

13 Applied Physiology, Nutrition, nd Metolism Pge 12 of Lfont, R. nd Dinn, L Prcticl uses for ecdysteroids in mmmls including humns: n updte. J. Insect Sci. 3: 7. PMID: Lplnte, M. nd Stini, D.M mtor signling in growth control nd disese. Cell 149(2): doi: /j.cell PMID: Molfino, A., Gioi, G., Rossi Fnelli, F., nd Muscritoli, M Bet-hydroxy-et- methylutyrte supplementtion in helth nd disese: systemtic review of rndomized trils Amino Acids 45(6): doi: /s z. PMID: Seidlov-Wuttke, D., Ehrhrdt, C., nd Wuttke, W Metolic effects of 20-OH-ecdysone in ovriectomized rts. J. Steroid Biochem. Mol. Biol. 119(3-5): doi: /j.jsm PMID: Toth, N., Hunydi, A., Bthori, M., nd Zdor, E Phytoecdysteroids nd vitmin D nlogues--similrities in structure nd mode of ction. Curr. Medicinl Chem. 17(18): doi: / #sthsh.hJDqr9m.dpuf. PMID: Toth, N., Szo, A., Kcsl, P., Heger, J., nd Zdor, E Hydroxyecdysone increses fier size in muscle-specific fshion in rt. Phytomed. Intl. J. Phytotherpy Phytophrmcol. 15(9): doi: /j.phymed PMID:

14 Pge 13 of 16 Applied Physiology, Nutrition, nd Metolism Wilorn, C.D., Tylor, L.W., Cmpell, B.I., Kerksick, C., Rsmussen, C.J., Greenwood, M., et l Effects of methoxyisoflvone, ecdysterone, nd sulfo-polyscchride supplementtion on trining dpttions in resistnce-trined mles. J. Intl. Soc. Sports Nutr. 3: doi: / PMID:

15 Applied Physiology, Nutrition, nd Metolism Pge 14 of Figure Legend Figure 1. Phosphoryltion of Akt, mtor nd 4E-BP1 in the skeletl muscle of mle rts orlly dministered 20HE. Phosphorylted Akt t threonine 308 nd totl Akt expression ws mesured y immunolot in Study 1 (A), Study 2 (D) nd Study 3 (G); Phosphorylted mtor t serine 2448 nd totl mtor ws mesured y immunolot in Study 1 (B), Study 2 (E) nd Study 3 (H); Gel moility shift ssy showing 4E-BP1 phosphoryltion in Study 1 (C), Study 2 (F), nd Study 3 (I). Br grphs represent mens ± SEM of densitometry rtios; n=5-6 per group. In Study 1 nd 3, time of euthnsi ws 30 min. In Study 3, 20HE dose ws 200 mg/kg nd leucine dose ws 1.35 g/kg. In ech r grph, mens not shring common letter re different, P< Figure 2. Phosphoryltion of Akt, mtor nd 4E-BP1 in the liver of mle rts orlly dministered 20HE Phosphorylted Akt t threonine 308 nd totl Akt expression ws mesured y immunolot in Study 1 (A), Study 2 (D) nd Study 3 (G); Phosphorylted mtor t serine 2448 nd totl mtor ws mesured y immunolot in Study 1 (B), Study 2 (E) nd Study 3 (H); Gel moility shift ssy showing 4E-BP1 phosphoryltion in Study 1 (C), Study 2 (F), nd Study 3 (I). Br grphs represent mens ± SEM of densitometry rtios; n=5-6 per group. In Study 1 nd 3, time of euthnsi ws 30 min. In Study 3, 20HE dose ws 200 mg/kg nd leucine dose ws 1.35 g/kg. In ech r grph, mens not shring common letter re different, P<

16 Pge 15 of 16 Applied Physiology, Nutrition, nd Metolism A. B. C. p-thr308 p-ser2448 Akt mtor mg/kg mg/kg g mg/kg D. E. F. p-thr308 p-ser2448 Akt hours mtor hours g hours G. H. I. p-thr308 p-ser2448 Akt mtor C L H LH C L H LH C L H LH g

17 Applied Physiology, Nutrition, nd Metolism Pge 16 of 16 A. B. C. p-thr308 p-ser2448 mtor Akt mg/kg mg/kg g mg/kg D. p-thr308 Akt hours E. F. p-ser2448 mtor hours g hours G. H. I. p-thr308 p-ser2448 mtor Akt C L H LH C L H LH C L H LH g