Regulation of protein degradation pathways by amino acids and insulin in skeletal muscle of neonatal pigs

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Surywn nd Dvis Journl of niml Science nd iotechnology 2014, 5:8 http://www.jssci.com/content/5/1/8 JOURNL OF NIML SCIENCE ND IOTECHNOLOGY RESERCH Open ccess Regultion of protein degrdtion pthwys y mino cids nd insulin in skeletl muscle of neontl pigs gus Surywn nd Teres Dvis * strct ckground: The rpid gin in len mss in neontes requires greter rtes of protein synthesis thn degrdtion. We previously delineted the moleculr mechnisms y which insulin nd mino cids, especilly leucine, modulte skeletl muscle protein synthesis nd how this chnges with development. In the current study, we identified mechnisms involved in protein degrdtion regultion. In experiment 1, 6- nd pigs were studied during 1) euinsulinemic-euglycemic-euminocidemic, 2) euinsulinemic-euglycemic-hyperminocidemic, nd 3) hyperinsulinemic-euglycemic-euminocidemic clmps for 2 h. In experiment 2, 5-d-old pigs were studied during 1) euinsulinemic-euglycemic-euminocidemic-euleucinemic, 2) euinsulinemic-euglycemic-hypominocidemichyperleucinemic, nd 3) euinsulinemic-euglycemic-euminocidemic-hyperleucinemic clmps for 24 h. We determined in muscle indices of uiquitin-protesome, i.e., trogin-1 (MFx) nd muscle RING-finger protein-1 (MuRF1) nd utophgy-lysosome systems, i.e., unc51-like kinse 1 (UKL1), microtuule-ssocited protein light chin 3 (LC3), nd lysosoml-ssocited memrne protein 2 (Lmp-2). For comprison, we mesured riosoml protein S6 (rps6) nd eukryotic initition fctor 4E (eif4e) ctivtion, components of trnsltion initition. Results: undnce of trogin-1, ut not MuRF1, ws greter in 26- thn pigs nd ws not ffected y insulin, mino cids, or leucine. undnce of ULK1 nd LC3 ws higher in younger pigs nd not ffected y tretment. The LC3-II/LC3-I rtio ws reduced nd ULK1 phosphoryltion incresed y insulin, mino cids, nd leucine. These responses were more profound in younger pigs. undnce of Lmp-2 ws not ffected y tretment or development. undnce of eif4e, ut not rps6, ws higher in 6- thn -pigs ut unffected y tretment. Phosphoryltion of eif4e ws not ffected y tretment, however, insulin, mino cids, nd leucine stimulted rps6 phosphoryltion, nd the responses decresed with development. Conclusions: The rpid growth of neontl muscle is in prt due to the positive lnce etween the ctivtion of protein synthesis nd degrdtion signling. Insulin, mino cids, nd, prticulrly, leucine, ct s signls to modulte muscle protein synthesis nd degrdtion in neontes. Keywords: mino cids, utophgy, Insulin, Leucine, Muscle, Neonte, Protein degrdtion, Protein synthesis, Swine, Uiquitin Introduction Skeletl muscle represents 40 50% of ody mss in mmmls nd is criticl regultor of overll metolism [1]. Therefore, n understnding of the processes involved in the postntl increse in muscle mss, with ssocited ccumultion of protein, is fundmentl [2]. We hve * Correspondence: tdvis@cm.edu United Sttes Deprtment of griculture/griculturl Reserch Service Children s Nutrition Reserch Center, Deprtment of Peditrics, ylor College of Medicine, Houston, TX 77030, US demonstrted tht protein deposition is high in skeletl muscle of neontl pigs nd decreses with ge [3]. This rpid gin in skeletl muscle mss in the neonte is in prt due the mrked increse in protein synthesis fter mel. The feeding-induced stimultion of muscle protein synthesis is independently regulted y the rise in insulin nd mino cids, especilly leucine [4,5]. lthough muscle protein deposition depends on the lnce etween the rtes of protein synthesis nd degrdtion, less is known 2014 Surywn nd Dvis; licensee iomed Centrl Ltd. This is n Open ccess rticle distriuted under the terms of the Cretive Commons ttriution License (http://cretivecommons.org/licenses/y/2.0), which permits unrestricted use, distriution, nd reproduction in ny medium, provided the originl work is properly cited. The Cretive Commons Pulic Domin Dediction wiver (http://cretivecommons.org/pulicdomin/zero/1.0/) pplies to the dt mde ville in this rticle, unless otherwise stted.

Surywn nd Dvis Journl of niml Science nd iotechnology 2014, 5:8 Pge 2 of 11 http://www.jssci.com/content/5/1/8 of the mechnisms tht regulte protein degrdtion in skeletl muscle of the neonte. The moleculr mechnisms y which insulin nd mino cids regulte protein synthesis hve een the suject of much investigtion [6,7]. Over the pst few yers, we [8,9] hve intensively studied the effect of the post-prndil rise in insulin nd mino cids on the ctivtion of signling components leding to protein synthesis in skeletl muscle of the neonte. We hve shown tht the stimultion of mmmlin trget of rpmycin (mtor) y insulin nd mino cids is enhnced in skeletl muscle of the neontl pig nd decreses with development [8]. ctivtion of mtor induces the phosphoryltion of 4E-inding protein 1 (4EP-1) nd riosoml protein S6K1 (S6K1), oth of which regulte mrn inding to the 43S pre-initition complex. However, the developmentl chnges in the response to mino cids nd insulin on the undnce nd the ctivtion of riosome protein S6 (rps6) nd eukryotic initition fctor 4E (eif4e) in skeletl muscle hs not een determined. Studies using in vitro nd in vivo methods indicte tht rps6 nd eif4e re crucil for the regultion of protein synthesis [10-13]. Of the mino cids, leucine is the most effective in cting s nutrient signl to ctivte protein synthesis in skeletl muscle of the neontl pig [14]. The uiquitin-protesome system (UPS) nd utophgylysosome system re mjor pthwys tht re involved in the regultion of protein degrdtion in skeletl muscle [15]. The utophgy-lysosome system plys significnt role in ulk proteolysis while the UPS is responsile for the control of the degrdtion of specific proteins [16]. UPS-dependent protein degrdtion is highly regulted [17]. In this system, lysyl residues of the trget proteins re serilly ttched y uiquitin ( 76-mino cid protein) which mrks them for protein degrdtion in the protesome. It is known tht two mjor muscle-specific E3 uiquitin ligses, MuRF1 (muscle RING-finger protein-1) nd trogin-1 (MFx), re importnt components of the UPS [17]. It hs ecome incresingly evident tht utophgy nd the UPS re needed for norml muscle development [18]. lthough in oth systems free mino cids cn e generted, only the utophgy system ppers to e physiologiclly regulted y mino cids [16]. utophgy is tightly regulted process tht involves the degrdtion of cell components including proteins through the lysosoml mchinery [19]. In norml physiologicl conditions, utophgy is ctive nd plys n importnt role in severl iologicl processes including cell development [20]. utophgy is crucil for the survivl of neontl nimls under strvtion conditions [21] nd is induced y erly wening in the piglet model [22]. In the lysosoml degrdtion pthwy, there re two mjor processes: mcroutophgy nd chperone-medited utophgy (CM). While the microtuule-ssocited protein 1 light chin 3 (LC3) is n importnt component or mrker for mcroutophgy, lysosome-ssocited memrne protein-2 (lmp-2) is crucil for CM processes [23,24]. mtor plys crucil role in the regultion of utophgy vi unc51-like kinse 1 (UKL1), n upstrem component of LC3 [25]. When the ctivtion of mtor is high, such s under nutrient sufficiency, mtor prevents the ctivtion of ULK1 y phosphorylting ULK1 t Ser 757 resulting in the suppression of utophgy [26]. Studies show tht oth insulin/igf-i (insulin-like growth fctor-i) nd mino cids regulte protein synthesis [6,7,27] nd protein rekdown [16,28], however, the role of mino cids on the ltter process is not well understood [29]. In vivo nd in vitro studies hve shown tht the rnched-chin mino cids, especilly leucine, ttenute muscle protein degrdtion [30,31]. However, the detiled moleculr spects of the mino cid-induced reduction of proteolysis in skeletl muscle through UPS nd utophgy hve not een elucidted [16]. The ojective of this study ws to determine the effects of the postprndil rise in mino cids nd insulin on the regultion of specific signling components involved in protein degrdtion, nd for comprison, protein synthesis, in skeletl muscle of neontl pigs nd how these re modulted y development. We further sought to identify the response of these intrcellulr signling components to more prolonged leucine dministrtion. To chieve this, 6- nd pigs were infused with mino cids or insulin to ttin post-prndil levels for 2 h in Experiment 1. In Experiment 2, 5-d-old pigs were infused for 24 h with physiologicl levels of leucine, without or with mino cid replcement to prevent leucine-induced hypominocidemi. Mteril nd methods nimls nd housing (experiment 1 nd 2) Multiprous cross-red (Lndrce Yorkshire Duroc Hmpshire) pregnnt sows (griculture Hedqurters, Texs Deprtment of Criminl Justice, Huntsville, TX) were housed in lcttion crtes in environmentlly controlled rooms prior to frrowing. Commercil diet (no. 5084; PMI Feeds, Richmond, IN) nd wter d liitum were provided. fter frrowing, piglets remined with the sow ut were not llowed ccess to the sow s diet. Three to four d efore study, minor surgery ws conducted to insert sterile ctheters into the jugulr vein nd crotid rtery [32]. The protocol ws pproved y the niml Cre nd Use Committee of ylor College of Medicine nd ws conducted in ccordnce with the Ntionl Reserch Council s Guide for the Cre nd Use of Lortory nimls.

Surywn nd Dvis Journl of niml Science nd iotechnology 2014, 5:8 Pge 3 of 11 http://www.jssci.com/content/5/1/8 Experimentl design Experiment 1 Piglets t 6 d of ge (1.9 ± 0.3 kg ody weight) nd t 26 d of ge (5.3 ± 0.8 kg ody weight), following n overnight fst, were rndomly ssigned to one of three tretment groups (n = 4 6 per tretment group): 1) euinsulinemic-euglycemic-euminocidemic conditions (C), 2) euinsulinemic-euglycemic-hyperminocidemic clmps (), nd 3) hyperinsulinemic-euglycemic-euminocidemic clmps (INS), s previously descried [33]. During the experiment, lood smples were collected nd immeditely nlyzed for glucose (YSI 2300 STT Plus; Yellow Springs Instruments, Yellow Springs, OH) nd totl rnched-chin mino cids (C) y rpid enzymtic kinetic ssy to estlish the sl concentrtions of lood glucose nd plsm rnched-chin mino cids to e used in the clmp technique. Clmps were initited with primed, constnt (12 ml/h) infusion of insulin (Eli Lilly, Indinpolis, IN) t 0 or 100 ng/kg 0.66 min given to ttin plsm insulin concentrtions of 3 (fsting insulin level) or 30 μu/ml (fed insulin level) nd sustined for period of 2 h. In order to clmp glucose nd mino cids t fsting levels, venous lood smples were otined every 5 min nd immeditely nlyzed for glucose nd C concentrtions. We djusted concentrtions within ± 10% of the sl fsting concentrtions. To otined euminocidemic conditions, the infusion rte of lnced mino cid mixture ws djusted to mintin plsm C within 10% of fsting levels. Likewise, hyperminocidemic conditions were otined y infusion of lnced mino cid mixture [34] to rise plsm C concentrtions y two-fold the fsting level to reproduce the level of mino cids present in the fed stte. To determine circulting insulin concentrtion, lood smples lso were tken t intervls. We chieved the desired sustrte nd hormone concentrtions trgetedduringourclmpproceduresdescriedinour previous puliction [33]. Plsm mino cids levels were rised two-fold to fed levels in the hyperminocidemic group nd plsm insulin levels were rised to the fed level (~30 μu/ml) in the hyperinsulinemic group. Circulting mino cids, insulin, nd glucose concentrtions levels were mintined t seline fsting levels during euminocidemi, euinsulinemi, nd euglycemi, respectively. Experiment 2 Overnight fsted 5-d-old piglets (2.6 ± 0.1 kg) were rndomly ssigned to one of three tretment groups (n =6/ group) nd studied during 1) euinsulinemic-euglycemiceuminocidemic-euleucinemic conditions (control, C), euinsulinemic-euglycemic-hypominocidemic-hyperleucinemic clmps (L), nd euinsulinemic-euglycemic-euminocidemic-hyperleucinemic (L + ) clmps for 24 h [35]. nimls ssigned to the C group were infused with sterile sline t 10 ml/h throughout the infusion period to chieve fsting levels of leucine. Piglets ssigned to the L group were infused with leucine t 400 μmol/kg h to rise circulting levels to tht of pigs fed high protein diet. Pigs in the L + group were infused with lncedminocidmixture[34],prepreddevoidof leucine, to mintin circulting mino cid concentrtions t seline fsting levels during the elevtion in leucine. The infusion rte of the mino cid mixture (devoid of leucine) ws progressively incresed t 10 min intervls from 0 to 0.4, 0.6, 0.85, 1.5, 1.85, 2.25, 2.7 nd 2.85 ml/kg h, until the infusion rte of 2.85 ml/kg h ws reched, nd mintined constnt throughout s previously clculted in our lortory [36]. We chieved the desired sustrte nd hormone concentrtions trgeted during our clmp procedure s descried in our previous puliction [35]. Immunolotting nd immunoprecipittion (experiment 1 nd 2) Frozen longissimus dorsi muscle smples were homogenized nd centrifuged t 10,000 g for 10 min t 4 C. The protein concentrtion ws determined in the superntnt y the rdford method [31]. Equl mounts (50 μg) of extrcted protein were electrophoreticlly seprted in polycrylmide gels nd trnsferred to polyvinylidene difluoride (PVDF) memrne (io-rd, Hercules, C), which were incuted with pproprite primry ntiodies followed y pproprite secondry ntiodies s previously descried [33]. lots were developed using n enhnced chemiluminescence kit (mershm), visulized, nd nlyzed using ChemiDoc-It Imging System (UVP, Uplnd, C). The protein undnce of ech signling components ws normlized with β-ctin undnce in the smples. Primry ntiodies tht were used in the immunolotting were MuRF1, trogin-1, β-ctin (Snt Cruz iotechnology, Snt Cruz, C), rps6, eif4e, Lmp-2, ULK1, nd LC3 (Cell Signling Technology, Dnvers, M). Sttisticl nlysis (experiment 1 nd 2) In Experiment 1, two-wy NOV with onferroni posttest were used to determine the effects of ge nd ech tretment nd their interction on the undnce nd phosphoryltion of protein degrdtion nd synthesis signling components. In Experiment 2, the sttisticl nlysis ws performed y one-wy NOV with susequent Tukey s post-test. Ech experiment used seprte control nimls. Proility vlues of P < 0.05 were considered sttisticlly significnt. Dt re presented s men ± SEM. Results We previously reported tht the undnce of mny positive regultors of protein synthesis ws significntly higher the younger the pig [33]. In this study, we extended

Surywn nd Dvis Journl of niml Science nd iotechnology 2014, 5:8 Pge 4 of 11 http://www.jssci.com/content/5/1/8 our nlysis to determine the effect of ge on the undnce nd the phosphoryltion of two dditionl positive regultors of protein synthesis (rps6 nd eif4e). s shown in Figure 1, the undnce of eif4e, ut not rps6, ws significntly higher in 6- compred to pigs (P < 0.05). s expected, short-term insulin or mino cid infusion hd no effect on eif4e or rps6 undnce. lthough neither insulin nor mino cids ltered the phosphoryltion of eif4e, insulin nd mino cids incresed the phosphoryltion of rps6 nd the response ws greter in 6- thn in pigs (P < 0.05) (Figure 2). Similr result ws otined on prolonged leucine infusion, where leucine, with or without mino cid replcement, hd no effect on eif4e phosphoryltion ut induced the phosphoryltion of rps6 (P < 0.05) (Figure 2). We determined the protein undnce of trogin-1 nd MuRF1 s indictors for the ctivtion of the uiquitin-protesome pthwy. s illustrted in Figure 3, the undnce of trogin-1 ws greter in 26- thn in 6- d-old-pigs (P < 0.05). Neither short-term insulin nor mino cid infusion, or more prolonged leucine infusion, hd n effect on the protein undnce of trogin-1 (Figure 3). With regrd to the undnce of the uiquitinprotesome component, MuRF1, there ws no effect of ge, cute mino cid or insulin infusion, or prolonged leucine dministrtion (Figure 4). To study the effect of mino cids nd insulin on the utophgy-lysosome system, we exmined ULK1, the LC3- II/LC3-I rtio ( commonly used mrker for utophgy) nd the lmp-2 undnce ( mrker for chperon-medited utophgy). First, we nlyzed the totl undnce nd phosphoryltion of ULK (Figure 5). ULK1 undnce ws higher in 6- thn in pigs (P < 0.05). cute mino cid or insulin infusion or more prolonged leucine dministrtion hd no effect on ULK1 undnce. mino cid- nd insulin-induced phosphoryltion of ULK1 ws lso higher in the younger pigs compred to their older counterprts (P < 0.05). Similrly, leucine infusion induced the phosphoryltion of ULK1 (P < 0.05). We found lso tht the totl undnce of LC3 decresed with ge (P < 0.05) (Figure 6). Insulin nd mino cids reduced the LC3-II/LC3-I rtio (P < 0.05), nd this effect eif4e undnce (U) eif4e undnce (U) C D rps6 undnce (U) rps6 undnce (U) C INS C INS C L L+ Figure 1 The protein undnce of eif4e nd rps6 in longissimus dorsi muscle in response to ge nd mino cids, insulin, nd leucine infusion. () Muscle eif4e undnce from experiment 1 during euinsulinemic-euglycemic-euminocidemic (control; C), euinsulinemic-euglycemic-hyperminocidemic (), nd hyperinsulinemic-euglycemic-euminocidemic (INS) clmps for 2 h in 6- nd pigs. () Muscle eif4e undnce from experiment 2 during euinsulinemic-euglycemic-euminocidemic-euleucinemic (C), euinsulinemiceuglycemic-hypominocidemic-hyperleucinemic (L), nd euinsulinemic-euglycemic-euminocidemic-hyperleucinemic (L+) clmps for 24 h in 5-d-old pigs. (C) Muscle rps6 undnce from experiment 1 during C,, nd INS clmps for 2 h in 6- nd pigs. (D) Muscle rps6 undnce from experiment 2 during C, L, nd L+ clmps for 24 h in 5-d-old pigs. Dt re normlized with β-ctin undnce nd re expressed in ritrry units (U). Vlues re mens ± SEM, n = 4 7. Vlues not shring common symols differ significntly (P < 0.05).

Surywn nd Dvis Journl of niml Science nd iotechnology 2014, 5:8 Pge 5 of 11 http://www.jssci.com/content/5/1/8 eif4e Phosphoryltion (U) eif4e Phosphoryltion (U) rps6 Phosphoryltion (U) C c C INS C INS C L L+ Figure 2 The phosphoryltion of eif4e nd rps6 in longissimus dorsi muscle in response to ge nd mino cids, insulin, nd leucine infusion. () Muscle eif4e phosphoryltion from experiment 1 during euinsulinemic-euglycemic-euminocidemic (control; C), euinsulinemiceuglycemic-hyperminocidemic (), nd hyperinsulinemic-euglycemic-euminocidemic (INS) clmps for 2 h in 6- nd pigs. () Muscle eif4e phosphoryltion from experiment 2 during euinsulinemic-euglycemic-euminocidemic-euleucinemic (C), euinsulinemic-euglycemichypominocidemic-hyperleucinemic (L), nd euinsulinemic-euglycemic-euminocidemic-hyperleucinemic (L+) clmps for 24 h in 5-d-old pigs. (C) Muscle rps6 phosphoryltion from experiment 1 during C,, nd INS clmps for 2 h in 6- nd pigs. (D) Muscle rps6 phosphoryltion from experiment 2 during C, L, nd L+ clmps for 24 h in 5-d-old pigs. Dt re normlized with eif4e nd rps6 undnce nd re expressed in ritrry units (U). Vlues re mens ± SEM, n = 4 7. Vlues not shring common symols differ significntly (P < 0.05). c rps6 Phosphoryltion (U) D ws greter in 6- thn in pigs (P < 0.05). Likewise, leucine infusion, with or without mino cid replcement, decresed the LC3-II/LC3-I rtio (P < 0.05). With respect to the undnce of Lmp-2, none of the tretments hd n effect on this component of the utophgy-lysosome system (Figure 7). Discussion In order to support their rpid growth, neontes hve higher protein turnover rtes thn dults. Since protein ccretion tkes plce when the rte of postprndil protein synthesis is higher thn the rte of protein degrdtion, the lnce etween these two processes is crucil. lthough the moleculr mechnisms y which protein synthesis nd protein degrdtion re governed hve een studied for while, most of reserch hs een performed in cell culture or hs focused on mture individuls [11,12,29,30]. Here we studied the effects of insulin nd mino cids, including leucine, on the undnce nd ctivtion of specific signling components of the protein synthesis nd degrdtion pprtus in neontl pigs nd how this chnges with development. Previously, we identified mjor signling components tht control protein synthesis in skeletl muscle of neontl pigs, however, modultion y rps6 nd eif4e ws not exmined [33]. Erly studies show tht rps6 phosphoryltion is involved in the regultion of glol protein synthesis [37]. More recent studies using the trnsgenic mouse model indicte tht rps6 is crucil for the regultion of cell size [38]. Other oservtions suggestthtfollowingmitogenicornutritionlsignls, rps6 is involved in n efficient mechnism for the ctivtion of protein synthesis tht ensures lnced protein synthesis nd controls energy wstge [39]. In the current study, we found tht the undnce of rps6 is not regulted y development. Likewise, neither mino cids nor insulin ffected rps6 undnce. Similr to its upstrem regultor, S6K1 [8,14], the phosphoryltion of rps6 ws stimulted y n cute infusion of insulin or mino cids tht reproduced the rise in

Surywn nd Dvis Journl of niml Science nd iotechnology 2014, 5:8 Pge 6 of 11 http://www.jssci.com/content/5/1/8 trogin-1 undnce (U) C INS C INS trogin-1 undnce (U) C L L+ Figure 3 The protein undnce of trogin-1 in longissimus dorsi muscle in response to ge nd mino cids, insulin, nd leucine infusion. () Muscle trogin-1 undnce from experiment 1 during euinsulinemic-euglycemic-euminocidemic (control; C), euinsulinemiceuglycemic-hyperminocidemic (), nd hyperinsulinemic-euglycemic-euminocidemic (INS) clmps for 2 h in 6- nd pigs. () Muscle trogin-1 undnce from experiment 2 during euinsulinemic-euglycemic-euminocidemic-euleucinemic (C), euinsulinemic-euglycemichypominocidemic-hyperleucinemic (L), nd euinsulinemic-euglycemic-euminocidemic-hyperleucinemic (L+) clmps for 24 h in 5-d-old pigs. Dt re normlized with β-ctin undnce nd re expressed in ritrry units (U). Vlues re mens ± SEM, n = 4 7. Vlues not shring common symols differ significntly (P < 0.05). these gents with feeding. The more prolonged dministrtion of leucine, in the presence of either hypominocidemi or euminocidemi, lso incresed rps6 phosphoryltion, indicting tht leucine ws primry driver for rps6 stimultion. The nolic effect of insulinndminocidswshigherin6-thnin26-dold pigs suggesting tht the ctivtion of rps6 my ply significnt role in the higher rte of protein synthesis during the neontl period. The trnsltion initition fctor, eif4e, is crucil for the regultion of cp-dependent trnsltion tht represents the stndrd mode of trnsltion used y the vst mjority of cellulr mrns [40]. Over- nd underexpression of eif4e indicte tht this initition fctor is importnt for the regultion of cell growth [41]. Furthermore, since eif4e is overexpressed in severl types of cncers, it is considered s primry trget for cncer drugs [42]. Interestingly, we found tht eif4e undnce is mrkedly elevted in 6- compred to pigs suggesting role of eif4e in the high muscle cell growth of young pigs. Studies lso show tht in severl types of cells, the phosphoryltion of eif4e, induced y nolic gents, is indispensle for protein synthesis nd cell growth [43,44]. In the current study, neither the ge nor nolic gent tretments hd ny effect on the phosphoryltion of eif4e. This finding is consistent with those of Vry et l. [13] who found tht the IGF-Iinduced stimultion of protein synthesis occurs in the sence of chnges in eif4e phosphoryltion. Nonetheless, the results show differentil regultion of the trnsltion initition signling proteins, rps6 nd eif4e, with development. While the undnce of eif4e decreses with ge, its phosphoryltion is unffected y the nolic gents tested. y contrst, the undnce of rps6 does not chnge with ge, ut its phosphoryltion in response to insulin nd mino cids decreses with development. The continul degrdtion nd synthesis of protein, i.e., protein turnover, is crucil for homeosttic functions of norml cells [45]. Studies show tht the uiquitinprotesome system plys mjor role in the regultion of muscle protein degrdtion [29]. The undnce of the muscle-specific uiquitin protein ligses (E3), trogin- 1/MFx nd MuRF1, is crucil for skeletl muscle degrdtion in ctolic sttes [46]. The trget protein sustrtes of trogin-1 include MyoD, trnscriptionl regultor which controls muscle size [46]. MuRF1 prefers structurl protein such s titin nd myosin light chin-1 (MLC1) s trget proteins [46]. Tken together, these ligses regulte the sustrte trgets tht ply n importnt role in skeletl muscle growth. In this study, we determined the undnce of these uiquitin ligses. We found tht only trogin-1 ws ffected y ge. lthough these results re consistent with the recent results of Orelln et l. [47], the differentil response of the two ligses seems inconsistent with their functions s mjor plyers of protein degrdtion. The finding tht these ligses re differentilly expressed in certin experimentl conditions is not uncommon. Frost et l. [48] found tht the sepsis-induced increse in trogin-1 mrn expression, ut not MuRF1, ws completely locked y IGF-I. Other studies show tht the trogin-1 mrn expression, ut not MuRF1, is increse y interleukin 6 (IL6) [49], nd ngiotensin II (NG II) [50]. Conversely, the skeletl muscle MuRF1 mrn expression, ut not trogin-1, is

Surywn nd Dvis Journl of niml Science nd iotechnology 2014, 5:8 Pge 7 of 11 http://www.jssci.com/content/5/1/8 MuRF1 undnce (U) C INS C INS MuRF1 undnce (U) C L L+ Figure 4 The protein undnce of MuRF1 in longissimus dorsi muscle in response to ge nd mino cids, insulin, nd leucine infusion. () Muscle MuRF1 undnce from experiment 1 during euinsulinemic-euglycemic-euminocidemic (control; C), euinsulinemiceuglycemic-hyperminocidemic (), nd hyperinsulinemic-euglycemic-euminocidemic (INS) clmps for 2 h in 6- nd pigs. () Muscle MuRF1 undnce from experiment 2 during euinsulinemic-euglycemic-euminocidemic-euleucinemic (C), euinsulinemiceuglycemic-hypominocidemic-hyperleucinemic (L), nd euinsulinemic-euglycemic-euminocidemic-hyperleucinemic (L+) clmps for 24 h in 5-d-old pigs. Dt re normlized with β-ctin undnce nd re expressed in ritrry units (U). Vlues re mens ± SEM, n = 4 7. Vlues not shring common symols differ significntly (P < 0.05). enhnced y exercise [46], inhiitor of nucler fctor kpp- kinsesuunitet(ikkβ) gene deletion [51], nd nucler fctor k (NFk) pthwy ctivtion [52]. lthough trogin-1 nd MuRF1 were discovered more thn decde go [53], their contriutions to the ctivtion of the uiquitin-protesome system re still controversil for severl resons [46]. First, their downstrem sustrtes re not similr nd in ddition to regulting protein degrdtion, they lso regulte other physiologicl functions [46]. Second, studies show tht their functions re species-specific. For exmple, the mrn expression of these ligses re incresed in old rts ut not in ged humns [54,55], nd fsting increses trogin-1 mrn expression in mice ut hs no effect in humns [56,57]. Lstly, studies suggest tht the ctivities of these ligses re ltered without chnges in their mrn expression levels nd tht the primry role of these ligses is not to enhnce muscle trophy [50]. Moreover, nother study showed tht lthough muscle protein degrdtion is elevted in helthy older humns when compred to their helthy younger counterprts, the mrn expression of these ligses is similr in oth ge groups [58]. Further studies re needed to evlute the functions of these uiquitin ligses nd whether they re relile mrkers for the uiquitin-protesome system in muscle [46]. Our oservtions lso show tht the undnce of these ligses ws not ffected y the cute rise in insulin or mino cids or more prolonged leucine dministrtion. One possile explntion of the lck of effect on these ligses is the short length of the fsting time used in this study (12 h). This my not hve een sufficiently long enough fsting time to enhnce the expression of these ligses nd thus the nimls my not hve een sufficiently ctolic for mino cids or insulin to reverse this ction. Severl rodent studies show tht prolonged fsting of 48 h or strvtion significntly incresed the mrn expression of these two ligses [59,60]. This suggests tht these uiquitin ligses do not ply criticl roles in the regultion of protein lnce during norml fsting-feeding cycles. utophgy plys significnt role in the ctolic process which is mnifested y the degrdtion of protein ggregtion nd dmged orgnelles vi the fusion etween utophgosomes nd lysosomes [18]. lthough mtor is crucil for the regultion of utophgy [61], the physiologicl role of utophgy in skeletl muscles hs not een fully elucidted. Studies show tht mtor inhiits utophgy vi inctivtion of ULK1, crucil component tht resides upstrem of LC3 [25]. We found tht the ULK1 undnce ws higher in younger pigs ut did not chnge with short-term insulin or mino cid infusion or more prolonged dministrtion of leucine. However, phosphoryltion of ULK1 t Ser 757 ws incresed in response to the cute rise in circulting insulin or mino cids, indicting mtor-induced inctivtion of ULK1, nd this effect ws reduced with development. Likewise, more sustined leucine dministrtion, in the presence of either hypominocidemi or euminocidemi, lso inctivted ULK1, likely y mtor-dependent mechnism. During fsting nd other stress conditions, utophgy hs the vitl role in mintining the mino cid pool y digesting musculr protein nd orgnelles [62]. In mcroutophgy, LC3 is widely known s mrker of utophgosomes [63]. Under utophgy-inducing conditions, LC3-I (the cytosolic form) is processed nd recruited to utophgosomes where the formtion of lipid conjugted LC3-II ttined y site specific proteolysis

Surywn nd Dvis Journl of niml Science nd iotechnology 2014, 5:8 Pge 8 of 11 http://www.jssci.com/content/5/1/8 Totl ULK1 (U) 0.8 0.6 0.4 0.2 Totl ULK1 (U) 1.0 0.8 0.6 0.4 0.2 0.0 0.0 C Phosphoryltion of ULK1 (U) 1.5 1.0 0.5 0.0 C INS C INS D C L L+ Figure 5 The undnce nd phosphoryltion of ULK1 in longissimus dorsi muscle in response to ge nd mino cids, insulin, nd leucine infusion. () Muscle ULK1 undnce from experiment 1 during euinsulinemic-euglycemic-euminocidemic (control; C), euinsulinemiceuglycemic-hyperminocidemic (), nd hyperinsulinemic-euglycemic-euminocidemic (INS) clmps for 2 h in 6- nd pigs. () Muscle ULK1 undnce from experiment 2 during euinsulinemic-euglycemic-euminocidemic-euleucinemic (C), euinsulinemic-euglycemichypominocidemic-hyperleucinemic (L), nd euinsulinemic-euglycemic-euminocidemic-hyperleucinemic (L+) clmps for 24 h in 5-d-old pigs. (C) Muscle ULK1 phosphoryltion from experiment 1 during C,, nd INS clmps for 2 h in 6- nd pigs. (D) Muscle ULK1 phosphoryltion from experiment 2 during C, L, nd L+ clmps for 24 h in 5-d-old pigs. Dt re expressed in ritrry units (U). Vlues re mens ± SEM, n = 4 7. Vlues not shring common symols differ significntly (P < 0.05). Phosphoryltion of ULK1 (U) 0.8 0.6 0.4 0.2 0.0 nd lipidtion ner the C-terminus occurs [63]. Since the formtion of LC3-II is positively correlted with utophgosome numers, mesuring the conversion of LC3-I to LC3-II y immunolotting is considered to e relile ssy to determine utophgic ctivity [63]. In the present study, short-term insulin or mino cid dministrtion or more prolonged leucine supplementtion suppressed the fsting-induced increse in skeletl muscle LC3-II protein levels. These nolic effects were more roust in younger pigs. Interestingly, we oserved decresed totl LC3 protein levels due to development indicting tht younger pigs hve higher sl utophgy. This oservtion is consistent with our previous findings of higher rtes of protein degrdtion in neontl thn in older rts [64]. We next sought to determine whether ge, the cute infusion of mino cids or insulin, or the chronic dministrtion of leucine ffect chperone-medited utophgy y mesuring the protein undnce of Lmp-2 [23,24]. We found tht the undnce of Lmp-2 ws not ffected y ny tretment. lthough genetic studies [65] hve shownthtlmp-2isinvolvedinregultingoverllutophgy in skeletl muscle, our dt suggest tht during the neontl period, chperone-medited utophgy does not ply mjor role in generl utophgy. Conclusions Throughout life, delicte lnce exists etween protein synthesis nd degrdtion tht is essentil for growth nd norml helth of humns nd nimls. Using neontl pigs, we hve sought to elucidte the moleculr mechnisms y which protein synthesis nd degrdtion re regulted, prticulrly in skeletl muscle. Hence, in these prticulr studies we focused on determining the effects of the cute rise in insulin nd mino cids, s well s the more prolonged dministrtion of leucine lone on the undnce nd ctivtion of signling components which re importnt plyers of protein degrdtion nd protein synthesis signling pthwys nd their modultion y development. Our results, sed on the ULK1 nd LC3 dt, nd the dt from Dvis et l. [64] showing development decline in the frctionl rte of protein degrdtion support the notion tht protein degrdtion ctivity is high during neontl period to mintin the elevted protein turnover needed to sustin growth. lthough the trogin-1 nd MuRF1 dt do not support this hypothesis, the regultion of these ligses is still controversil nd, thus more studies re needed. The results, long with previous findings [66], further suggest tht eif4e nd rps6 ply crucil roles in ensuring

Surywn nd Dvis Journl of niml Science nd iotechnology 2014, 5:8 Pge 9 of 11 http://www.jssci.com/content/5/1/8 Totl LC3 (U) Totl LC3 (U) C LC3-II/LC3-I rtio (U) c D LC3-II/LC3-I rtio (U) C INS C INS C L L+ Figure 6 The undnce of totl LC3 nd the rtio of LC3-II to LC3-I in longissimus dorsi muscle in response to ge nd mino cids, insulin, nd leucine infusion. () Muscle LC3 undnce from experiment 1 during euinsulinemic-euglycemic-euminocidemic (control; C), euinsulinemic-euglycemic-hyperminocidemic (), nd hyperinsulinemic-euglycemic-euminocidemic (INS) clmps for 2 h in 6- nd pigs. () Muscle LC3 undnce from experiment 2 during euinsulinemic-euglycemic-euminocidemic-euleucinemic (C), euinsulinemiceuglycemic-hypominocidemic-hyperleucinemic (L), nd euinsulinemic-euglycemic-euminocidemic-hyperleucinemic (L+) clmps for 24 h in 5-d-old pigs. (C) Muscle LC3-II to LC3-I rtio from experiment 1 during C,, nd INS clmps for 2 h in 6- nd pigs. (D) Muscle LC3-II to LC3-I rtio from experiment 2 during C, L, nd L+ clmps for 24 h in 5-d-old pigs. Dt re expressed in ritrry units (U). Vlues re mens ± SEM, n = 4 7. Vlues not shring common symols differ significntly (P < 0.05). Lmp -2 undnce (U) Lmp -2 undnce (U) C INS C INS C L L+ Figure 7 The protein undnce of Lmp-2 in longissimus dorsi muscle in response to ge nd mino cids, insulin, nd leucine infusion. () Muscle Lmp-2 undnce from experiment 1 during euinsulinemic-euglycemic-euminocidemic (control; C), euinsulinemiceuglycemic-hyperminocidemic (), nd hyperinsulinemic-euglycemic-euminocidemic (INS) clmps for 2 h in 6- nd pigs. () Muscle Lmp-2 undnce from experiment 2 during euinsulinemic-euglycemic-euminocidemic-euleucinemic (C), euinsulinemic-euglycemichypominocidemic-hyperleucinemic (L), nd euinsulinemic-euglycemic-euminocidemic-hyperleucinemic (L+) clmps for 24 h in 5-d-old pigs. Dt re normlized with β-ctin undnce nd re expressed in ritrry units (U). Vlues re mens ± SEM, n = 4 7.

Surywn nd Dvis Journl of niml Science nd iotechnology 2014, 5:8 Pge 10 of 11 http://www.jssci.com/content/5/1/8 high rtes of protein synthesis in skeletl muscle of neontl pigs. With respect to utophgy, the cute rise in insulin nd mino cids, similr to tht which occurs with feeding, s well s the more prolonged supplementtion with leucine lone, irregrdless of the circulting levels of other mino cids, hd inhiitory effects on ULK1 nd LC3-II. These responses re consistent with the suppressive effects of ULK1 nd LC3-II on protein degrdtion. Likewise, ll tretments hd positive effect on the phosphoryltion of rps6, ut not eif4e, indicting tht stimultion of eif4e phosphoryltion is not crucil for nolic-induced ctivtion of mrn trnsltion in skeletl muscle. Understnding how protein synthesis nd protein degrdtion re regulted during the neontl period is crucil for the development of new nutritionl strtegies tht cn support mximum growth of neontes. revitions 4EP-1: 4E-inding protein 1; NG II: ngiotensin II; trogin-1: muscle-specific F-ox protein highly expressed during muscle trophy; C: rnched-chin mino cids; CM: Chperone-medited utophgy; eif4e: Eukryotic initition fctor 4E; IGF-I: Insulin-like growth fctor-1; IKKβ: Inhiitor of nucler fctor kpp- kinse suunit et; IL6: Interleukin 6; Lmp-2: Lysosoml-ssocited memrne protein 2; LC3: Microtuule-ssocited protein light chin 3; NFk: Nucler fctor k; mtor: Mmmlin trget of rpmycin; MuRF1: Muscle RING-finger protein-1; rps6: Riosoml protein S6; S6K1: Riosoml protein S6 kinse 1; ULK1: Unc51-like kinse 1; UPS: Uiquitin-protesome system. Competing interests The uthors declre tht they hve no competing interests. uthors contriutions S nd TD did the conception nd design of the reserch; S performed the experiments; S nlyzed the dt; S nd TD interpreted the results of the experiments; S prepred the figures nd drfted the mnuscript; S nd TD edited nd revised the mnuscript; nd TD hd primry responsiility for the finl content. oth uthors pproved the finl version of the mnuscript. cknowledgements This work is puliction of the US Deprtment of griculture/griculturl Reserch Service (USD/RS) Children s Nutrition Reserch Center, Deprtment of Peditrics, ylor College of Medicine. This project hs een funded in prt y Ntionl Institute of rthritis nd Musculoskeletl nd Skin Diseses Grnt R-044474 (T.. Dvis), Ntionl Institute of Child Helth nd Humn Development HD-072891 (T.. Dvis), nd y the USD/RS under Coopertive greement no. 6250-510000-055 (T.. Dvis). The contents of this puliction do not necessrily reflect the views or policies of the US Deprtment of griculture, nor does the mention of trde nmes, commercil products, or orgniztions imply endorsement y the US Government. Received: 8 Septemer 2013 ccepted: 14 Jnury 2014 Pulished: 17 Jnury 2014 References 1. Sndri M: utophgy in skeletl muscle. FES Lett 2010, 584:1411 1416. 2. Young VR: Regultion of protein synthesis nd skeletl muscle growth. J nim Sci 1974, 38:1054 1070. 3. Dvis T, urrin DG, Fiorotto ML, Nguyen HV: Protein synthesis in skeletl muscle nd jejunum is more responsive to feeding in 7-thn in 26-dyold pigs. m J Physiol 1996, 270:E802 E809. 4. O Connor PM, ush J, Surywn, Nguyen HV, Dvis T: Insulin nd mino cids independently stimulte skeletl muscle protein synthesis in neontl pigs. m J Physiol Endocrinol Met 2003, 284:E110 E119. 5. 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