Adrenaline-stimulated glycogen breakdown activates glycogen synthase and. increases insulin-stimulated glucose uptake in epitrochlearis muscles

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1 Articles in PresS. Am J Physiol Endocrinol Met (Decemer 2, 214). doi:1.1152/jpendo Adrenline-stimulted glycogen rekdown ctivtes glycogen synthse nd increses insulin-stimulted glucose uptke in epitrochleris muscles Anders J. Kolnes 1, Jesper B. Birk 2, Einr Eilertsen 3, Jorid T. Stuenæs 3, Jørgen F. P. Wojtszewski 2, Jørgen Jensen 2, Chrles University Third Fculty of Medicine, Prgue, Czech Repulic 2 Moleculr Physiology Group, The August Krogh Centre, Deprtment of Nutrition, Exercise nd Sports, Copenhgen University, Copenhgen, Denmrk 3 Ntionl Institute of Occuptionl Helth, Oslo, Norwy 4 Deprtment of Physicl Performnce, Norwegin School of Sports Sciences, P.O.Box 414 Ullevål Stdion, Sognsveien 22, NO-86 Oslo, Norwy Running Title: Cori cycle nd insulin sensitivity in skeletl muscles Corresponding Author: Jørgen Jensen, Deprtment of Physicl Performnce, Norwegin School of Sport Sciences, Sognsvnn 22, P.O.Box 414 Ullevål Stdion, 86 Oslo, Norwy Phone: Fx: E.mil: jorgen.jensen@nih.no Copyright 214 y the Americn Physiologicl Society.

2 ABSTRACT Adrenline increses glycogen synthse (GS) phosphoryltion nd decreses GS ctivity ut lso stimultes glycogen rekdown nd low glycogen content normlly ctivtes GS. To test the hypothesis tht glycogen content directly regultes GS phosphoryltion, glycogen rekdown ws stimulted in condition with decresed GS ctivtion. Sline or drenline (.2mg/1g rt) ws injected sucutneously in Wistr rts (~13 g) with low (24 h fsted), norml (norml diet) nd high glycogen content (fsted-refed) nd epitrochleris muscles were removed fter 3 h nd incuted ex vivo eliminting drenline ction. Adrenline injection reduced glycogen content in epitrochleris muscles with high (12.7±17.8 vs 24.6±14.5 mmol kg -1 ; p<.1) nd norml glycogen (89.5±7.6 vs 152±8.1 mmol kg -1 ; p<.1), ut not significntly in muscles with low glycogen (9.±5. vs 12.8±7.8 mmol kg - 1 ; p=.17). In sline-injected rts, GS phosphoryltion t sites 2+2, 3+3 nd 1 ws higher nd GS ctivity lower in muscles with high compred to low glycogen. GS site 2+2 nd 3+3 phosphoryltion decresed nd GS ctivity incresed in muscles where drenline decresed glycogen content; these prmeters were unchnged in epitrochleris from fsted rts where drenline injection did not decrese glycogen content. Incution with insulin decresed GS site 3+3 phosphoryltion independent of glycogen content. Insulin-stimulted glucose uptke ws incresed in muscles where drenline injection decresed glycogen content. In conclusion, drenline stimultes glycogenolysis in epitrochleris muscles with norml nd high, ut not low glycogen content. Adrenline-stimulted glycogenolysis decresed GS phosphoryltion nd incresed GS ctivity. These dt for the first time document direct regultion of GS phosphoryltion y glycogen content Keywords: Insulin sensitivity, GSK3, AMPK, TBC1D4, Dietes, phosphoryltion, liver, Cori cycle 2

3 INTRODUCTION Skeletl muscle glycogen is n importnt energy sustrte during exercise (24). In ddition, skeletl muscle glycogen functions s mjor store for ingested crohydrtes nd contriutes to regultion of lood glucose (21). The glycogen content in skeletl muscles is limited nd high glycogen content inhiits glycogen synthse (GS) ctivity wheres low glycogen content ctivtes glycogen synthse (9; 19; 29; 33). This inverse reltionship etween glycogen content nd glycogen synthse ctivtion hs een estlished y mnipulting glycogen content y fsting-refeeding (19; 28-3) nd exercise-diet protocols (33). Adrenline stimultes glycogen rekdown in skeletl muscles s muscle contrction does, ut drenline lso increses GS phosphoryltion nd decreses GS ctivity (5; 18; 38). The effect of drenline-stimulted glycogen rekdown on GS phosphoryltion fter removl of drenline hs not een crefully chrcterised. Glycogen synthse is phosphorylted t t lest 9 sites nd incresed phosphoryltion decreses frctionl ctivity (5; 2; 36). Adrenline stimultes GS phosphoryltion vi β 2 - drenergic receptors, camp nd ctivtion of PKA; ctivted PKA phosphoryltes GS t sites 2, 1 nd 1 (5; 36). In ddition, PKA increses GS phosphoryltion vi phosphoryltion of the protein phosphtse-1 (PP1) glycogen trgeting regultory suunit R GL. Phosphoryltion of R GL dissocites PP1 from the glycogen prticle nd increses GS phosphoryltion (36). Incresed glycogen content is ssocited with incresed phosphoryltion wheres decresed glycogen content decreses GS phosphoryltion t severl sites (19; 25; 29; 35). Insulin increses GS ctivity vi PI3-kinse nd reduces phosphoryltion of GS t sites phosphorylted y GSK-3 (site 3, 3, 3c nd 4) (2; 2; 36). Muscle contrction ctivtes GS independent of GSK-3, ut requires R GL to ctivte GS (1). Reduced glycogen content fter exercise contriutes to increse GS ctivity (33), ut muscle contrction dditionlly increses GS ctivity independent of glycogen content (29). PP1 ctivity hs een suggested to decrese s glycogen content increses nd llow GS phosphoryltion (2; 36). Fvouring this ide, it hs een shown tht overexpression of R GL decreses GS phosphoryltion nd increses glycogen content wheres the glycogen content is low in muscles where R GL is deleted (1). Glycogen content nd muscle contrction re much stronger regultors of GS phosphoryltion nd ctivity thn insulin (9; 19; 33), which my reflect more ctive phosphtse ctivity nd comprehensive dephosphoryltion of GS thn insulin-stimulted inhiition of GSK-3. 3

4 Three compnying ppers entitled: The mechnism of epinephrine ction (6-8) re the experimentl ckground for the Cori cycle stting tht glycogen moilized in the muscles is converted into liver glycogen with lctic cid s n intermediry stge. (p. 317 in Cori & Cori, 1928). The three ppers consisted of experiments where drenline ws injected into rts under different nutritionl sttus nd it ws shown tht drenline directed glycogen to the liver t the expense of crcss glycogen. Importntly, the effect of drenlinestimulted glycogen rekdown on GS phosphoryltion nd ctivtion fter removl of drenline hs not een crefully chrcterised. The primry im of the present study ws to investigte GS phosphoryltion nd ctivtion in epitrochleris muscles following drenlinestimulted glycogen rekdown nd fter removl of drenline. The studies were performed in muscles with different glycogen content to investigte whether initil glycogen content influences the mgnitude of drenline-stimulted glycogen rekdown nd GS ctivtion. We hypothesized tht skeletl muscle GS phosphoryltion nd ctivtion would dpt to the new glycogen content vi uto-regultory feedck mechnisms fter removl of drenline. The glycogen content in skeletl muscles influences insulin-, contrction- nd phrmcologicl stimulted glucose uptkes. In rodents, fsting nd refeeding (17; 19; 29; 3), cute exercise (1; 11) nd trining (26) hve een used to modulte glycogen content, nd estlished n inverse reltionship etween glycogen content nd glucose uptke. In humns, glucose uptke is lso regulted y glycogen when glycogen content is modulted y exercise nd diet (42). There is lso evidence tht decresing glycogen content y drenline stimultion increses insulin-stimulted glucose uptke (22; 34). However, these studies do not llow seprting the effect of drenline-stimulted glycogen rekdown from potentil effects independent of glycogen content. The secondry im ws therefore to investigte the role of drenline-medited glycogen rekdown on insulin-stimulted glucose uptke in muscles with different glycogen content. MATERIALS AND METHODS Mle Wistr rts were kept on 12/12 h light/drk cycles t 21 C nd 55 % humidity with free ccess to chow nd wter for t lest one week efore experiments. Prior to experiments, glycogen content in epitrochleris muscle ws mnipulted y fsting nd refeeding s descried previously (17). Briefly, rts with low glycogen (LG) were fsted for 24 h prior to experiments. Rts with norml glycogen (NG) were kept on their norml diet until 4

5 experiments. Rts with high glycogen (HG) were fsted for 24 h followed 24 h with free ccess to chow prior to experiments (Figure 1). Fsting ws strted t 9. A.M. On experimentl dys, rts were given sucutneous injection of sline (.9 % sline) or drenline (.2 mg per 1 g ody weight of rts), nd plced lone in new cges without food ut ccess to wter. Adrenline (Epinephrine (+) itrtrte, E4375, Sigm, St Louis, MO) ws dissolved in.9 % sline (.1 mg/ml), nd.2 ml/1 g odyweight ws injected sucutneously in the ck of the rts. Muscles used for incution were removed 3 h fter sline/drenline injection. Additionl muscles nd livers were removed nd frozen directly in liquid nitrogen from group of rts without injection nd with free ccess to food nd wter until experiments (untreted controls) nd from group of rts 1 h fter drenline/sline injection. Blood glucose ws lso mesured in these rts. The weight of the rts ws g on the dy of experiment. Rts were nesthetized with n intrperitonel injection of 7.5 mg of sodium pentoritl (5 mg/ml) per 1 g rt 3 h fter drenline/sline injection. Blood smples were tken from the til for mesured glucose (HemoCue AB, Ängelholm, Sweden) fter nesthetistion. Epitrochleris nd soleus muscles were dissected for incution. Additionl muscles (dt not included in this rticle) were dissected out nd frozen in liquid nitrogen. Finlly, the liver ws dissected out, weighed nd piece ws frozen in liquid nitrogen. Muscles nd liver tissue were stored t 7 C until nlyses. The experiments nd procedures were pproved y Ntionl Animl Reserch Authority nd performed ccording to lws nd regultions controlling experiments on live nimls in Norwy, nd the Europen Convention for the Protection of Verterte Animls used in Experimentl nd Other Scientific Purposes. Incutions: Epitrochleris muscles were immeditely mounted on holders t their pproximte resting length nd preincuted 45 min in 3.5 ml Kres-Henseleit uffer contining 5.5 mm glucose, 2 mm pyruvte,.1 % ovine serum lumin (Frction V), nd 5 mm HEPES s descried (27). The incutions were performed t 3 ºC nd the uffers were gssed with 95 % O 2 /5 % CO 2. After preincution, glucose uptke ws mesured during 3 min s descried previously (27). In rief,.25 µci/ml 2-[1,2 3 H(N)]-deoxy-D-glucose (25.5 Ci/mmol; DuPont, NEN) nd.1 µci/ml [1-14 C]-D-mnnitol (51.5 mci/mmol; DuPont, NEN) were dded to the uffer (contining 5.5 mm glucose) nd glucose uptke ws clculted from the intrcellulr ccumultion of [ 3 H]-2-deoxy-D-glucose s descried previously (Aslesen nd Jensen, 1998). Insulin (Actrpid, Novo Nordisk) ws dded in concentrtions of 1 µu/ml (physiologicl 5

6 concentrtion) or 1, µu/ml s indicted in figures. Muscles were, fter the 3 min incution, rpidly removed from the holders voiding unnecessry stretching, lotted on filter pper nd frozen in liquid nitrogen. For mesurement of glucose uptke nd glycogen, out hlf the muscle ws freeze-dried nd weighed nd dissolved in 6 µl 1 M KOH for 2 min t 7 C. Of the digest, 4 µl ws dded to 3 ml scintilltion solution (Hionic-Fluor, Pckrd), mixed nd counted for rdioctivity (TRI-CARB 46C, Pckrd, USA). Glycogen content in muscles. For nlysis of glycogen, 1 l of the KOH digest ws cidified with 25 l 7 M cetic cid, efore 5 l.3 M cette uffer (ph 4.8) contining.2 U/ml of myloglucosidse (Boehringer-Mnnheim) ws dded, nd the glycogen ws hydrolysed t 37 C for 3 hours. Glucose units were mesured enzymticlly with rections (hexokinse nd glucose 6-phosphte dehydrogense) coupled to NADPH formtion s descried y Lowry nd Pssonneu (32). Rections were llowed for 3 min t room temperture in 96 well pltes, nd fluorescence ws mesured on BMG plte reder (BMG, Germny). Glycogen content in liver: Liver smples were freeze-dried, weighed nd glycogen ws hydrolysed in 1 M HCl (2.5 h t 1 C). Hydrolyste ws neutrlised with NOH s descried (23). Homogenistion of muscles: Epitrochleris muscle tissue (~ 1 mg) ws homogenised in uffer contining 5 mm HEPES (ph 7.5), 1 % glycerol, 2 mm N-pyrophosphte, 15 mm NCl, 1 % NP-4, 2 mm β-glycerolphosphte, 1 mm NF, 2 mm PMSF, 1 mm EDTA, 1 mm EGTA, 1 µg l -1 protinin, 1 µg l -1 leupeptin, 2 mm N 3 VO 4, 3 mm enzmidine. Homogentes were rotted for 1 h t 4 C. Protein content ws nlysed in triplictes in 96-well plte with the icinchoninic cid method (Pierce Chemicl Co, Rockford, IL). GS ctivity. Glycogen synthse ctivity ws mesured with the filter pper method nd 14 C- UDP-glucose (Perkin Elmer) s sustrte s descried y Thoms et l. (37), ut modified to 96-well filter pltes (Unifilter 35 pltes, Wtmn, Cmridge, UK) s descried (15). Glycogen synthse ctivity ws mesured with 1.5 mm UDP-glucose (High) nd.3 mm UDP-glucose (physiologicl) in ssy uffer nd different concentrtions of glucose 6- phosphte. Totl GS ctivity ws mesured with 8 mm glucose 6-phosphte. GS ctivity mesured with.17 mm glucose 6-phosphte ws used to clculte frctionl velocity [FV = (ctivity with.17 mm G6P) / (ctivity with 8 mm G6P)]. GS ctivity mesured with.1 6

7 mm glucose 6-phosphte ws used to clculte GS I-form [I-form = (ctivity with.1 mm G6P) / (ctivity with 8 mm G6P)]. Western lot. Proteins were seprted using 1 % SDS gels nd trnsferred (semidry) to polyvinyl difluoride (PVDF) memrnes (Immoilion Trnsfer Memrne, Millipore AS, Copenhgen, Denmrk). Memrnes were proed with ntiodies ginst GS site 2+2, GS site 3+3, nd GS site 1 s descried (15). The ntiodies used to study phosphoryltion of GSK-3β, AMPK, nd TBC1D4 were: GKS-3β (BD Trnsduction Lortories) GSK3 α/β Ser 21/9 (Cell Signling Technology, Dnvers, MA), AMPK Thr 172 (Cell Signling Technology), AMPK-α2 (Snt Cruz Biotechnology Inc, Dlls, TX), TBC1D4 (Millipore, Billeric, MA) nd TBC1D1 Thr 642 (Symnsis, New Zelnd). Sttistics: Dt re presented s mens ± SEM. Anlysis of vrince ws performed to investigte differences nd Fisher s lest significnt difference (LSD) ws used s post hoc test to compre different tretments. Liver glycogen content vried ~75-fold etween fsted nd fsted-refed rts, nd effect of drenline injection on liver glycogen content ws compred with T-test within similr feeding protocol. P<.5 ws considered s significnt. RESULTS Blood glucose ws ~4 mm in 24 h fsted rts, nd lood glucose incresed to ~8 mm during drenline injection (Figure 2A). Blood glucose ws ~8 mm in rts on norml diet nd in fsted-refed rts. It peked t ~14 mm 1 h fter drenline injection ut styed stle during sline injection (Figure 2B-C). Three hours fter drenline injection lood glucose remined slightly higher in drenline-injected rts compred to sline-injected rts. Liver glycogen content in rts on norml diet, ut fsted for 3 h fter sline injection, ws ~1 mmol kg dw -1 nd drenline injection did not influence glycogen content (Tle 1). Liver glycogen content ws reduced to very low levels during 24 h fsting, nd drenline injection incresed glycogen content y ~ 1 mmol kg dw -1 (p<.1; Tle 1). Fsting-refeding incresed liver glycogen content to ~ 13 mmol kg dw -1 nd drenline injection did not influence glycogen content (Tle 1). Glycogen content in epitrochleris muscles from rts on norml diet ws ~15 mmol kg dw -1 3 h fter sline infusion, wheres glycogen content 3 h fter drenline infusion ws decresed to ~1 mmol kg dw -1 (Figure 3A). In epitrochleris from fsted-refed rts the 7

8 glycogen content ws higher thn in epitrochleris from rts kept on norml diet nd ws ~2 mmol/kg dw 3 h fter sline infusion. Three h fter drenline injection glycogen content ws reduced to ~12 mmol/kg dw in epitrochleris muscles from fsted-refed rts nd glycogen content tended to e higher thn in muscles from rts on norml diet injected with drenline (p<.1). In epitrochleris muscles from fsted rts glycogen contents ws ~1 mmol kg dw -1 nd drenline injection did not decrese glycogen content significntly (Figure 3A). Glycogen synthse ctivity ws influenced y glycogen content in sline-injected rts, oth when mesured with physiologicl nd high concentrtion of UDP-glucose (Figure 3B- E). Glycogen synthse frctionl ctivities nd GS I-form-% mesured with.3 mm UDPglucose (FV.3 nd I-form.3 ) were, s expected, much lower thn ctivities mesured with 1.5 mm UDP-glucose, ut the effect of glycogen content ws comprle. Glycogen synthse ctivity ws incresed in muscles from drenline-injected rts on norml diet nd fstedrefed (Figure 3B-E). In epitrochleris muscles from fsted rts, glycogen synthse ctivity ws not incresed in muscles from drenline-injected rts; of note, drenline did not decrese glycogen content in these muscles. Some epitrochleris muscles were removed 1 h fter drenline injection nd frozen immeditely to descrie the cute effect of drenline injection; drenline injection decresed GS ctivity s expected in ll groups lthough not significntly in epitrochleris from fsted-refed rts (Tle 2). Phosphoryltion of GS site 2+2 nd GS site 3+3 were influenced y modultion of glycogen content y fsting nd fsting-refeeding (Figure 3F-G). GS phosphoryltion t site 2+2 nd site 3+3 ws reduced in epitrochleris muscles from drenline-injected rts on norml diet nd fsted-refed (Figure 3F,G); drenline injection decresed glycogen content in muscles from these groups of rts (Figure 3A). GS site 1 phosphoryltion ws higher in epitrochleris from fsted-refed rts compred to muscles from fsted rts (Figure 3H). GS site 1 ws not regulted y drenline injection (Figure 3H). GSK-3β Ser 9 phosphoryltion ws not influenced y glycogen content or drenline injection (Figure 3I). AMPK Thr 172 phosphoryltion ws higher in epitrochleris from fsted rts compred to oth rts on norml diet nd from fsted-refed rts (Figure 3J). AMPK Thr 172 phosphoryltion ws similr in muscles from sline- nd drenline-injected rts (Figure 3J). Insulin incresed GS ctivtion in epitrochleris from sline-injected rts (Figure 4). However, insulin-stimulted GS ctivtion ws minly oserved when GS ctivities were 8

9 mesured with physiologicl concentrtion of UDP-glucose. GS FV.3 nd GS I-form.3 were incresed y insulin in epitrochleris from rts on norml diet nd form fsted rts injected with sline (Figure 4G-H, J-K). Insulin only incresed GS GS I-form 1.5 in epitrochleris muscles from rts on norml diet (Figure 4B), nd insulin did not increse FV 1.5 significntly in ny groups (Figure 4D-F). In epitrochleris muscles from drenline-injected rts, insulin incresed GS ctivtion in ll groups independent of glycogen content when mesured with.3 mm UDP-glucose (Figure 4G-L). With high concentrtion of UDPglucose, insulin incresed GS I-form % in epitrochleris from drenline-injected rts on norml diet (Figure 4B). Insulin did not influence GS site 2+2 phosphoryltion in ny of the groups (Figure 5A-C). Insulin decresed GS site 3+3 phosphoryltion in epitrochleris muscles with low nd norml glycogen, ut not in epitrochleris from fsted-refed rts injected with sline (Figure 5 D-F). GS site 3+3 phosphoryltion ws lower in epitrochleris from drenlineinjected in ll groups lso during insulin stimultion except in muscles from fsted rts in the presence of 1, µu/ml insulin (5D). In epitrochleris muscles from drenline-injected rts on norml diet or fsted rts, insulin stimultion tended to decrese GS site 3+3 phosphoryltion (p<.1; Figure 5D-E). Insulin did not decrese GS site 3+3 phosphoryltion in drenline-injected epitrochleris muscles from fsted-refed rts (Figure 5F). GS site 1 phosphoryltion ws not regulted y insulin (Figure 5G-I). Insulin incresed GSK-3β Ser 9 phosphoryltion similrly in epitrochleris muscles from fsted, fsted-refed nd rts on norml diet independent of drenline injection (Figure 5J-L). Incution with insulin did not influence AMPK Thr 172 phosphoryltion (Dt not shown). Bsl nd insulin-stimulted glucose uptke ws higher in epitrochleris muscles from fsted rts thn in muscles from rts on norml diet nd fsted-refed rts when sline ws injected. Insulin incresed glucose uptke in ll groups (Figure 6B,E,H). Insulin-stimulted glucose uptke ws higher in epitrochleris muscles from drenline-injected rts on norml diet nd fsted-refeed rts (Figure 6E,H); drenline injection decresed glycogen content in these muscles (Figure 6D,G). In muscles from fsted rts, glucose uptke ws similr in sline- nd drenline-injected rts when mesured with (sl) nd 1, µu/ml of insulin (Figure 6B). Glucose uptke ws higher in epitrochleris muscles from drenlineinjected mesured with 1 µu/ml of insulin (Figure 6B). Of note, glycogen content ws 9

10 lower in epitrochleris muscles from fsted rts fter drenline injection t this insulin concentrtion (Figure 6A). Insulin incresed TBC1D4 Thr 642 phosphoryltion in muscles independent of glycogen content (Figure 6C,F,I). Furthermore, insulin-stimulted TBC1D4 Thr 642 phosphoryltion ws similr in muscles from sline- nd drenline-injected rts despite tht glucose uptke ws higher when glycogen content ws reduced y drenline injection DISCUSSION It is well documented tht glycogen content influences GS phosphoryltion nd ctivity, ut the mechnisms linking glycogen content to regultion of GS ctivity remin elusive. Most previous studies hve mnipulted glycogen content y comintion of exercise nd diet or fsting-refeeding protocols. Therefore, to further understnd the role of glycogen content on GS phosphoryltion nd ctivtion, drenline-stimulted glycogen rekdown ws initited in muscles with different glycogen content to test the hypothesis tht GS phosphoryltion dpts to reduced glycogen content when drenline is removed. GS phosphoryltion ws decresed nd GS ctivtion incresed in epitrochleris muscles with drenlin-stimulted glycogen rekdown. Importntly, drenline did not decrese glycogen content in muscles with low glycogen content nd no effect on GS phosphoryltion or ctivtion ws oserved in these muscles showing condition during which drenline stimultion per se does not modulte GS phosphoryltion nd ctivtion. The primry im of the present study ws to investigte the effect of drenlinemedited glycogen rekdown on GS phosphoryltion nd GS ctivity. Adrenline ctivtes PKA, nd the kinse phosphoryltes GS t sites 2, 1 nd 1 (5; 18; 36; 38). PKA lso phosphoryltes the PP1 inding protein R GL, which releses PP1 from the glycogen prticle nd increses GS phosphoryltion (4). The present study ws designed to stimulte glycogen rekdown in conditions where glycogen synthse ws highly phosphorylted nd inctivted, nd our dt confirmed tht GS ctivity ws reduced in the muscles immeditely frozen 1 h fter drenline injection s expected (9; 18; 38). The min finding in the present study ws tht GS phosphoryltion t site 2+2 nd site 3+3 ws reduced fter removl of drenline in muscles where drenline injection reduced glycogen content. GSK-3β phosphoryltes GS site 3, 3, 3c nd 4 (36) ut GSK-3β phosphoryltion ws not regulted y drenline injection or y glycogen content. We hve previously shown tht when glycogen content is mnipulted y fsting-refeding, GS 1

11 phosphoryltion is regulted independent of GSK-3β (19). Indeed, reduction in glycogen content y fsting requires drenline (41) nd our dt show tht oth fsting nd drenline injection decresed GS phosphoryltion independent of GSK-3. Insulin, on the other hnd, requires GSK-3 phosphoryltion for GS ctivtion (2). In the present study, insulin incresed GSK-3β Ser 9 phosphoryltion nd decresed GS site 3+3 phosphoryltion, ut not phosphoryltion of GS site 2+2, greeing with previous studies in rt skeletl muscles (13; 16; 19; 29-31). The insulin-medited increse in GS ctivity ws lso less thn the increse in muscles where glycogen content ws reduced y drenline, s expected (9; 19; 29; 33). AMPK phosphoryltes GS site 2 (25; 36). However, AMPK phosphoryltion ws similr in muscles from sline- nd drenline-injected rts, nd the lower GS site 2+2 phosphoryltion in muscles from drenline-injected rts occurred without regultion of AMPK phosphoryltion. Muscle contrction decreses GS phosphoryltion t site 2+2 nd site 3+3 (29; 3; 35), s we oserved in muscles where drenline injection decresed glycogen content. Muscle contrction requires R GL for ctivtion of GS suggesting tht contrction regultes GS phosphoryltion vi PP1 ctivtion (1). This grees with the more generl dephosphoryltion of GS during muscle contrction compred to insulin stimultion (3). Muscles with high glycogen hve reduced protein phosphtse ctivity wheres overexpression of R GL increses glycogen content (1) supporting the concept tht glycogen content regultes GS phosphoryltion vi PP1. Indeed, drenline-stimulted glycogen rekdown did not decrese GS site 1 phosphoryltion, ut overll, our dt support tht drenline-stimulted glycogen content decreses GS phosphoryltion vi ctivtion of PP1. We suggest tht R GL ecomes dephosphorylted fter removl of drenline nd recruits PP1 ck to the glycogen prticle nd dephosphoryltes GS in reltion to the glycogen content. This is the first documenttion tht drenline-medited glycogen rekdown is directly linked to regultion of GS phosphoryltion nd ctivtion. Indeed, Dnforth (9) used drenline in one of his experiments to modulte glycogen content to study the reltionship etween glycogen content nd GS ctivtion, ut it is not possile to seprte effects independent of reduced glycogen content in tht study. The dt in the present study enles us to seprte the effect of glycogen content from tht of drenline stimultion per se s drenline did not significntly reduce glycogen content in muscles from fsted rts. In muscles from fsted rts drenline did not influence GS phosphoryltion nd GS ctivtion. Therefore, the present study for the first time documents direct uto-regultion of GS phosphoryltion y glycogen content. 11

12 The modultion of glycogen content in epitrochleris muscles prior to drenline injection lso showed tht the glycogen content regultes drenline-stimulted glycogenolysis. In greement with previous study (34) drenline injection decresed muscle glycogen content y 45 % in muscle from rts on norml diet. Adrenline did not significntly decrese glycogen content in epitrochleris from fsted rts suggesting tht muscles with low glycogen content re protected from complete depletion y the stress hormone. Previously, glycogen phosphorylse ctivtion ws reported to e lower in glycogen-depleted muscles nd increses s glycogen stores re repleted (3; 12). Glycogen phosphorylse ctivtion nd glycogenolytic rte re lso elevted during muscles contrction in muscles with high glycogen (14). Although we did not find significnt reduction in glycogen content in epitrochleris from fsted rts (~1% lower), lood glucose incresed fter drenline injection nd liver glycogen incresed s reported y Cori nd Cori (6), showing tht drenline reloctes crohydrtes etween orgns. Insulin-stimulted glucose uptke ws incresed in muscles where drenline injection reduced glycogen content, greeing with previous studies in which glycogen content ws modulted y fsting-refeeding or exercise nd diet (1; 17; 19). An incresed insulinstimulted glucose uptke fter drenline injection hs een reported previously (22; 34), ut this is the first comprehensive investigtion where drenline ws injected in rts with different glycogen content. In epitrochleris from rts on norml diet nd from fsted-refed rts, where drenline reduced glycogen content, glucose uptke ws incresed t oth physiologicl nd high insulin concentrtion indicting tht oth insulin sensitivity nd responsiveness were incresed. Of note, glucose uptke t physiologicl insulin concentrtion ws incresed in muscles from fsted rts, ut these muscles lso hd smll reduction in glycogen content, nd insulin-stimulted glucose uptke ws only incresed in muscles where drenline stimulted glycogen rekdown. Insulin incresed TBC1D4 Thr 642 phosphoryltion s expected (4; 39). Importntly, insulin-stimulted TBC1D4 Thr 642 phosphoryltion ws similr in muscles from sline- nd drenline-injected rts despite tht insulin-stimulted glucose ws elevted in muscles where drenline decresed glycogen content. Therefore, drenline-stimulted glycogen content did not influence TBC1D4 Thr 642 nd GSK-3β Ser 9 phosphoryltion indicting tht insulin signlling ws not mplified t key molecules regulting glucose uptke nd GS ctivity, respectively. The present dt therefore support mechnistic link etween glycogen content nd stimultion of glucose uptke, lthough such mechnism remins to e documented. 12

13 In conclusion, drenline stimulted glycogen rekdown in epitrochleris muscles with norml nd high glycogen content ut not in muscles with low glycogen content. GS phosphoryltion decresed nd GS ctivtion incresed in muscles where drenline injection decresed glycogen content. Importntly, drenline did not decrese glycogen content in muscles from fsted rts nd GS ws neither dephosphorylted nor ctivted in these muscles. Therefore, our dt for the first time documents direct uto-regultion of glycogen synthse phosphoryltion y glycogen content fter removl of drenline. Our dt lso show tht drenline-stimulted glycogen rekdown increses insulin-stimulted glucose uptke nd therefore tht the Cori cycle contriutes to incresed insulin sensitivity in skeletl muscles. Acknowledgement We thnk Betin Bolmgren, Astrid Bolling, Ad Ingvldsen, Fng-Chin Lin, Dvid Håkensen, nd Kristoffer Cumming for technicl ssistnce. Professor Erik A Richter is thnked for most useful comments to the mnuscript. This work ws crried out s prt of the progrm of the UNIK: Food, Fitness & Phrm for Helth nd Disese (see The UNIK project is supported y the Dnish Ministry of Science, Technology nd Innovtion. The study ws funded y the Dnish Council for Independent Reserch Medicl Sciences (FSS), the Novo Nordisk Foundtion. The Dnish Dietes Acdemy supported the study vi Visiting Professorship to JJ t Deprtment of Nutrition, Exercise nd Sports, Copenhgen University. 13

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17 Jensen J, Ruzzin J, Jeens E, Brennesvik EO nd Knrdhl S. Improved insulin-stimulted glucose uptke nd glycogen synthse ctivtion in rt skeletl muscles fter drenline infusion: role of glycogen content nd PKB phosphoryltion. Act Physiol Scnd 184: , Jensen J, Tntiwong P, Stuenes JT, Molin-Crrion M, DeFronzo RA, Skmoto K nd Musi N. Effect of cute exercise on glycogen synthse in muscle from oese nd dietic sujects. Am J Physiol Endocrinol Met 33: E82-E89, Jensen TE nd Richter EA. Regultion of glucose nd glycogen metolism during nd fter exercise. J Physiol 59: , Jørgensen SB, Nielsen JN, Birk JB, Olsen GS, Viollet B, Andreelli F, Schjerling P, Vulont S, Hrdie DG, Hnsen BF, Richter EA nd Wojtszewski JF. The lph2-5'amp-ctivted protein kinse is site 2 glycogen synthse kinse in skeletl muscle nd is responsive to glucose loding. Dietes 53: , Kwnk K, Hn DH, Nolte LA, Hnsen PA, Nktni A nd Holloszy JO. Decresed insulin- stimulted GLUT-4 trnsloction in glycogen-supercompensted muscles of exercised rts. Am J Physiol 276: E97-E912, Kolnes AJ, Ingvldsen A, Bolling A, Stuenes JT, Kreft M, Zorec R, Shepherd PR nd Jensen J. Cffeine nd theophylline lock insulin-stimulted glucose uptke nd PKB phosphoryltion in rt skeletl muscles. Act Physiol (Oxf) 2: 65-74,

18 Li YC, Lin FC nd Jensen J. Glycogen content regultes insulin- ut not contrction-medited glycogen synthse ctivtion in the rt slow-twitch soleus muscles. Act Physiol (Oxf) 197: , Li YC, Stuenæs JT, Kuo CH nd Jensen J. Glycogen content nd contrction regulte glycogen synthse phosphoryltion nd ffinity for UDP-glucose in rt skeletl muscles. Am J Physiol Endocrinol Met 293: E1622-E1629, Li YC, Zrrinpshneh E nd Jensen J. Additive effect of contrction nd insulin on glucose uptke nd glycogen synthse in muscle with different glycogen contents. J Appl Physiol 18: , Lin FC, Bolling A, Stuenes JT, Cumming KT, Ingvldsen A, Li YC, Ivy JL nd Jensen J. Effect of insulin nd contrction on glycogen synthse phosphoryltion nd kinetic properties in epitrochleris muscles from len nd oese Zucker rts. Am J Physiol Cell Physiol 32: C1539- C1547, Lowry OH nd Pssonneu JV. A flexile system of enzymtic nlysis. New York: Acdemic Press, 1972, p. p Nielsen JN, Derve W, Kristinsen S, Rlston E, Ploug T nd Richter EA. Glycogen synthse locliztion nd ctivity in rt skeletl muscle is strongly dependent on glycogen content. J Physiol 531: , Nolte LA, Gulve EA nd Holloszy JO. Epinephrine-induced in vivo muscle glycogen depletion enhnces insulin sensitivity of glucose trnsport. J Appl Physiol 76: ,

19 Prts C, Helge JW, Nordy P, Qvortrup K, Ploug T, Del F nd Wojtszewski JF. Dul regultion of muscle glycogen synthse during exercise y ctivtion nd comprtmentliztion. J Biol Chem 284: , Roch PJ, DePoli-Roch AA, Hurley TD nd Tglircci VS. Glycogen nd its metolism: some new developments nd old themes. Biochem J 441: , Thoms JA, Schlender KK nd Lrner J. A rpid filter pper ssy for UDPglucose-glycogen glucosyltrnsferse, including n improved iosynthesis of UDP- 14 C-glucose. Anl Biochem 25: , Toole BJ nd Cohen PT. The skeletl muscle-specific glycogen-trgeted protein phosphtse 1 plys mjor role in the regultion of glycogen metolism y drenline in vivo. Cell Signl 19: , Treek JT, Pehmoller C, Kristensen JM, Kjosted R, Birk JB, Schjerling P, Richter EA, Goodyer LJ nd Wojtszewski JF. Acute exercise nd physiologicl insulin induce distinct phosphoryltion signtures on TBC1D1 nd TBC1D4 proteins in humn skeletl muscle. J Physiol 592: , Wlker KS, Wtt PW nd Cohen P. Phosphoryltion of the skeletl muscle glycogen- trgetting suunit of protein phosphtse 1 in response to drenline in vivo. FEBS Lett 466: , Winder WW nd Dun C. Control of Fructose 2,6-Diphosphte in Muscle of Exercising Fsted Rts. Am J Physiol 262: E919-E924,

20 Wojtszewski JF, McDonld C, Nielsen JN, Hellsten Y, Hrdie DG, Kemp BE, Kiens B nd Richter EA. Regultion of 5'AMP-ctivted protein kinse ctivity nd sustrte utiliztion in exercising humn skeletl muscle. Am J Physiol Endocrinol Met 284: E813-E822,

21 LEGENDS Figure 1. Schemtic overview of the study design Figure 2. Blood glucose concentrtion fter injection of drenline or sline in rts under different nutritionl conditions. Blood smples were tken from groups of rts efore injection nd 1 or 3 h fter sucutneous injection of drenline (.2 mg/1 g rt) or sline. (A) Blood glucose concentrtion in rts fsted for 24 h efore nd fter injection of sline (open symols) or drenline (filled symols). (B) Blood glucose concentrtion in rts mintined on their norml diet until injection of sline (open symols) or drenline (filled symols). (C) Blood glucose concentrtion in fsted-refed rts efore nd fter injection of sline (open symols) or drenline (filled symols). All groups were fsted fter injection of drenline or sline. For ll groups: Before injection ( h) N=8; 1 h fter injection: n=4; 3 h fter injection: n=14. : p<.5 compred to sline Figure 3. Effect of drenline injection on glycogen content, glycogen synthse ctivtion nd phosphoryltion of glycogen synthse, GSK-3β nd AMPK in epitrochleris muscles from rts under different nutritionl conditions. Rt were diet mnipulted s descried in Methods. Sline or drenline (.2 mg/1 g rt) ws injected sucutneously nd epitrochleris muscles were removed 3 h lter nd pre-incuted for 45 min nd incuted 3 min in control uffer. (A) Glycogen content in epitrochleris muscles fter injection of sline (open rs) or drenline (filled symols). (B) Glycogen synthse % I-form 1.5 (mesured with 1.5 mm UDP-glucose) in epitrochleris muscles fter injection of sline (open rs) or drenline (filled symols). (C) Glycogen synthse FV 1.5 (mesured with 1.5 mm UDPglucose) in epitrochleris muscles fter injection of sline (open rs) or drenline (filled symols). (D) Glycogen synthse % I-form.3 (mesured with.3 mm UDP-glucose) in epitrochleris muscles fter injection of sline (open rs) or drenline (filled symols). (E) Glycogen synthse FV.3 (mesured with.3 mm UDP-glucose) in epitrochleris muscles fter injection of sline (open rs) or drenline (filled symols). (F) Glycogen synthse site 2+2 phosphoryltion in epitrochleris muscles fter injection of sline (open rs) or drenline (filled symols). (G) Glycogen synthse site 3+3 phosphoryltion in epitrochleris muscles fter injection of sline (open rs) or drenline (filled symols). (H) Glycogen synthse site 1 phosphoryltion in epitrochleris muscles fter injection of sline 21

22 (open rs) or drenline (filled symols). (I) GSK-3β Ser 9 phosphoryltion in epitrochleris muscles fter injection of sline (open rs) or drenline (filled symols). (J) AMPK Thr 172 phosphoryltion in epitrochleris muscles fter injection of sline (open rs) or drenline (filled symols). Dt re Mens ± SEM; n=7-8 in ll groups. : p<.5; (): p<.1 compred to muscles from sline-treted rts; : p<.5; (): p<.1 compred to muscles from fsted rts treted similrly; c: p<.5 compred to norml diet with similr tretment. Figure 4. Effect of drenline injection on insulin-stimulted glycogen synthse ctivtion in epitrochleris muscles from rts under different nutritionl conditions. Rt were diet mnipulted s descried nd sline or drenline (.2 mg/1 g rt) ws injected sucutneously nd epitrochleris muscles were removed 3 h lter nd pre-incuted for 45 min nd incuted 3 min in uffer without nd with different concentrtions of insulin. (A- C) Glycogen synthse % I-form 1.5 in epitrochleris muscles from 24 h fsted rts (A), rts on norml diet (B) nd fsted-refed rts (C) fter injection of sline (open rs) or drenline (filled symols) nd incuted with different concentrtions of insulin (, 1 µu/ml, 1, µu/ml). (D-F) Glycogen synthse FV 1.5 in epitrochleris muscles from 24 h fsted rts (D), rts on norml diet (E) nd fsted-refed rts (F) fter injection of sline (open rs) or drenline (filled symols) nd incuted with different concentrtions of insulin (, 1 µu/ml, 1, µu/ml). (G-I) Glycogen synthse % I-form.3 in epitrochleris muscles from 24 h fsted rts (G), rts on norml diet (H) nd fsted-refed rts (I) fter injection of sline (open rs) or drenline (filled symols) nd incuted with different concentrtions of insulin (, 1 µu/ml, 1, µu/ml). (J-L) Glycogen synthse FV.3 in epitrochleris muscles from 24 h fsted rts (J), rts on norml diet (K) nd fsted-refed rts (L) fter injection of sline (open rs) or drenline (filled symols) nd incuted with different concentrtions of insulin (, 1 µu/ml, 1, µu/ml). Dt re Mens ± SEM; n=7-8 in ll groups. : p<.5 compred to muscles from sline-treted rts; : p<.5 compred to muscles from fsted rts treted similrly; c: p<.5 compred to norml diet with similr tretment Figure 5. Effect of drenline injection on insulin-stimulted phosphoryltion of glycogen synthse nd GSK-3 β in epitrochleris muscles from rts under different nutritionl conditions. Rt were diet mnipulted s descried nd sline or drenline (.2 mg/1 g rt) ws injected sucutneously nd epitrochleris muscles were removed 3 h lter nd pre- 22

23 incuted for 45 min nd incuted 3 min in uffer without nd with different concentrtions of insulin. (A-C) Glycogen synthse Site 2+2 phosphoryltion in epitrochleris muscles from 24 h fsted rts (A), rts on norml diet (B) nd fsted-refed rts (C) fter injection of sline (open rs) or drenline (filled symols) nd incuted with different concentrtions of insulin (, 1 µu/ml, 1, µu/ml). (D-F) Glycogen synthse Site 3+3 phosphoryltion in epitrochleris muscles from 24 h fsted rts (D), rts on norml diet (E) nd fsted-refed rts (F) fter injection of sline (open rs) or drenline (filled symols) nd incuted with different concentrtions of insulin (, 1 µu/ml, 1, µu/ml). (G-I) Glycogen synthse Site 1 phosphoryltion in epitrochleris muscles from 24 h fsted rts (G), rts on norml diet (H) nd fsted-refed rts (I) fter injection of sline (open rs) or drenline (filled symols) nd incuted with different concentrtions of insulin (, 1 µu/ml, 1, µu/ml). (J-L) GSK-3β Ser 9 phosphoryltion in epitrochleris muscles from 24 h fsted rts (J), rts on norml diet (K) nd fsted-refed rts (L) fter injection of sline (open rs) or drenline (filled symols) nd incuted with different concentrtions of insulin (, 1 µu/ml, 1, µu/ml). Dt re Mens ± SEM; n=7-8 in ll groups. : p<.5; ():p<.1 compred to muscles from sline-treted rts; : p<.5; (): p<.1 compred to muscles from fsted rts treted similrly; c: p<.5; (c): p<.1 compred to norml diet with similr tretment. Figure 6. Effect of drenline injection on glycogen content, insulin-stimulted glucose uptke nd TBC1D4 phosphoryltion in epitrochleris muscles from sline- nd drenlinetreted rts under different nutritionl conditions. Adrenline/sline ws injected 3 h efore muscles were dissected out for incution. (A) Glycogen content in epitrochleris muscles from 24 h fsted rts fter injection of sline (open rs) or drenline (filled symols). Glycogen content ws mesured fter 3 min incution with, 1 µu/ml, 1, µu/ml of insulin. (B) Bsl nd insulin-stimulted glucose uptke in epitrochleris muscles from 24 h fsted rts fter injection of sline (open rs) or drenline (filled symols). Glucose uptke ws mesured during 3 min incution with, 1 µu/ml, 1, µu/ml of insulin. (C) Bsl nd insulin-stimulted TBC1D4 Thr 642 phosphoryltion in epitrochleris muscles from 24 h fsted rts fter injection of sline (open rs) or drenline (filled symols). TBC1D4 Thr 642 phosphoryltion ws mesured fter 3 min incution with, 1 µu/ml, 1, µu/ml of insulin. (D) Glycogen content in epitrochleris muscles from rts on norml diet fter injection of sline (open rs) or drenline (filled symols). (E) Bsl nd insulinstimulted glucose uptke in epitrochleris muscles from rts on norml diet fter injection of 23

24 sline (open rs) or drenline (filled symols). (F) Bsl nd insulin-stimulted TBC1D4 Thr 642 phosphoryltion in epitrochleris muscles from rts on norml diet fter injection of sline (open rs) or drenline (filled symols). (G) Glycogen content in epitrochleris muscles from fsted-refed rts fter injection of sline (open rs) or drenline (filled symols). (H) Bsl nd insulin-stimulted glucose uptke in epitrochleris muscles from fsted-refed rts fter injection of sline (open rs) or drenline (filled symols). (I) Bsl nd insulin-stimulted TBC1D4 Thr 642 phosphoryltion in epitrochleris muscles from fstedrefed rts fter injection of sline (open rs) or drenline (filled symols). Dt re Mens ± SEM; n=7-8 in ll groups. : p<.5 compred to muscles from sline-treted rts; : p<.5; (): p<.1 compred to muscles from fsted rts treted similrly; c: p<.5; compred to norml diet with similr tretment

25 Tle 1. Effect of drenline injection on liver glycogen content in rts under different nutritionl conditions. Rts were injected with sline or drenline (.2 mg/1 g rt) nd livers were removed 3 h lter Glycogen (mmol/kg dw) Fsted Norml diet Fsted/Refed Sline 17.8±1.1 (9) 978.1±45.7 (16) ±55.7 (16) c Adrenline 12.±23.3 (9) 964.2±43.9 (16) ±64.5 (16) c Dt re Men ± SEM. Glycogen contents (mmol kg dw -1 ) in livers from rts without injection were: Fsted: 26.2±2.9 (7); Norml diet ±77.5 (8); Fsted-Refed ±73.9 (8). Groups compred with ANOVA; T-test ws used for comprison of effect of drenline. : p<.1 compred to sline. : p<.1 compred to Fsted. c: p<.1 compred to Norml diet. Tle 2. Glycogen synthse I-form in epitrochleris muscles directly frozen 1 h fter drenline injection. Adrenline ws injected sucutneously nd muscles were removed 1 h lter nd GS I-form 1.5 (mesured with 1.5 mm UDP glucose). Glycogen synthse I-Form 1.5 (%) Control Sline 1 h Adrenline 1 h Fsted 53.1± ± ±1.7 Norml diet 51.3± ± ±4.5 Fsted-refed 39.3±4.1 c 37.5±9.3 c 25.2±8.7 Dt re men ±SEM. : p<.5 compred to Sline; : p<.5 compred to Control. c: p<.5 compred to Fsted nd Norml diet. Control: n=7-8; Sline nd Adrenline 1 h: n=4 for ll groups

26 Figure 1

27 Figure 2 A B C Blood glucose (mm) Fsted-Sline Fsted-Adrenline Time (h) Blood glucose (mm) Norml diet-sline Norml diet-adrenline Time (h) Blood glucose (mm) Ftsted/Refed-Sline Fsted/Refed-Adrenline Time (h)