ARTICLE. Abstract Aims/hypothesis The adiponectin signalling pathway is largely unknown, but recently the adaptor protein containing

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1 Dibetologi (211) 54: DOI 1.17/s x ARTICLE Incresed bundnce of the dptor protein contining pleckstrin homology domin, phosphotyrosine binding domin nd leucine zipper motif (APPL1) in ptients with obesity nd type 2 dibetes: evidence for ltered diponectin signlling R. M. Holmes & Z. Yi & E. De Filippis & R. Berri & S. Shhni & P. Sthynryn & V. Shermn & K. Fujiwr & C. Meyer & C. Christ-Roberts & H. Hwng & J. Finlyson & L. Q. Dong & L. J. Mndrino & M. Bjj Received: 2 Jnury 211 /Accepted: 29 Mrch 211 /Published online: 12 My 211 # The Author(s) 211. This rticle is published with open ccess t Springerlink.com Abstrct Aims/hypothesis The diponectin signlling pthwy is lrgely unknown, but recently the dptor protein contining pleckstrin homology domin, phosphotyrosine binding domin nd leucine zipper motif (APPL1), hs been shown to interct directly with diponectin receptor (ADIPOR)1. APPL1 is present in C2C12 myoblsts nd mouse skeletl muscle, but its presence in humn skeletl muscle hs not been investigted. Methods Smples from type 2 dibetic, nd len nd nondibetic obese prticipnts were nlysed by: immunoprecipittion nd western blot; HPLC-electrospry ionistion (ESI)-mss spectrometry (MS) nlysis; pek re nlysis by MS; HPLC-ESI-MS/MS/MS nlysis; nd RT-PCR nlysis of APPL1 mrna. Results Immunoprecipittion nd western blot indicted bnd specific to APPL1. Tryptic digestion nd HPLC-ESI- MS nlysis of whole-muscle homogente APPL1 unmbiguously identified APPL1 with 56% sequence coverge. Pek re nlysis by MS vlidted western blot results, showing APPL1 levels to be significntly incresed in type 2 dibetic nd obese s compred with len prticipnts. Electronic supplementry mteril The online version of this rticle (doi:1.17/s x) contins supplementry mteril, which is vilble to uthorised users. R. M. Holmes Deprtment of Physiology, University of Texs Helth Science Center, Sn Antonio, TX, USA L. Q. Dong Deprtment of Cellulr nd Structurl Biology, University of Texs Helth Science Center, Sn Antonio, TX, USA L. Q. Dong Deprtment of Phrmcology, University of Texs Helth Science Center, Sn Antonio, TX, USA E. De Filippis : R. Berri : K. Fujiwr Deprtment of Medicine, University of Texs Helth Science Center, Sn Antonio, TX, USA R. M. Holmes : Z. Yi : E. De Filippis : K. Fujiwr : C. Meyer : C. Christ-Roberts : H. Hwng : J. Finlyson : L. J. Mndrino (*) Center for Metbolic nd Vsculr Biology, College of Liberl Arts nd Sciences, PO Box 87374, Tempe, AZ , USA e-mil: Lwrence.mndrino@su.edu L. J. Mndrino Deprtment of Medicine, Myo Clinic-Myo Clinic Arizon, 134 Est She Boulevrd, Scottsdle, AZ 85259, USA S. Shhni : P. Sthynryn : V. Shermn : M. Bjj (*) Endocrinology Division, Bylor College of Medicine nd St Luke s Hospitl, 179 Dryden Street, Houston, TX 773, USA e-mil: mndeepbjj@hotmil.com

2 Dibetologi (211) 54: Trgeted phosphopeptide nlysis by HPLC-ESI-MS/MS/ MS showed tht APPL1 ws phosphorylted specificlly on Ser 41. APPL1 mrna expression ws significntly incresed in obese nd type 2 dibetic prticipnts s compred with len prticipnts. After britric surgery in morbidly obese prticipnts with subsequent weight loss, skeletl muscle APPL1 bundnce ws significntly reduced (p<.5) in ssocition with n increse in plsm diponectin (p<.1), incresed levels of ADIPOR1 (p<.5) nd incresed muscle AMP-ctivted protein kinse (AMPK) phosphoryltion (p<.5). Conclusions/interprettion APPL1 bundnce is significntly higher in type 2 dibetic muscle; APPL1 is phosphorylted in vivo on Ser 41. Improvements in hyperglycemi nd hypodiponectinemi following weight loss re ssocited with reduced skeletl muscle APPL1, nd incresed plsm diponectin levels nd muscle AMPK phosphoryltion. Keywords Adiponectin. APPL1. Obesity. Type 2 dibetes Abbrevitions ADIPOR Adiponectin receptor AMPK AMP-ctivted protein kinse APPL1 Adptor protein contining pleckstrin homology domin, phosphotyrosine binding domin nd leucine zipper motif ESI Electrospry ionistion-mss MS Mss spectrometry m/z Mss:chrge rtio p38mapk p38 Mitogen-ctivted protein kinse Introduction The dipocyte hormone, diponectin, hs recently received much ttention due to its insulin-sensitising effects on lipid nd glucose metbolism [1 3]. The moleculr cuses of insulin resistnce re currently unknown, but one possible mechnism is ltered or impired diponectin signlling. Defects in diponectin signlling cn rise from ltertions in totl diponectin concentrtions, defects in diponectin receptor (ADIPOR) levels or defects in intrcellulr signlling molecules. Low serum concentrtions of diponectin ccompny insulin resistnce [4] nd hve been ssocited with disorders such s obesity [5 7], hyperlipidemi [8, 9], crdiovsculr disese [1] nd type 2 dibetes [11]. mrna of both ADIPORs (ADIPOR1, ADIPOR2) is highly expressed in humn skeletl muscle [12], s opposed to mouse, where Adipor2 is most bundntly expressed in liver nd Adipor1 is predominntly expressed in myotubes [13]. The intrcellulr diponectin signlling pthwy is still lrgely unknown, despite the identifiction of two ADI- PORs, ADIPOR1 nd ADIPOR2 [13]. ADIPORs re seven trnsmembrne domin receptors, but re unique from G-protein coupled receptors due to their opposite loction of the intrcellulr N-terminl nd the extrcellulr C- terminl [13]. Insulin-resistnt ob/ob mice hve decresed bundnce of ADIPOR1 nd ADIPOR2 [14]; but mrna expression of ADIPOR1 nd ADIPOR2 showed no difference between helthy, len control nd obese type 2 dibetic individuls [15]. However, Jng et l. reported tht ADIPOR2 levels re significntly lower in type 2 dibetic prticipnts thn in len controls, with ADIPOR1 following the sme trend, lbeit without sttisticl significnce [16]. ADIPOR1 nd ADIPOR2 mrna expression is significntly decresed in norml glucose-tolernt individuls with strong fmily history of dibetes s compred with those with no history of dibetes [12]. In ddition, insulin-stimulted glucose disposl is positively ssocited with ADIPOR1 nd ADIPOR2 expression [12]. Finlly, cultured myotubes from obese controls nd obese dibetic prticipnts showed incresed levels of ADIPOR1 reltive to len controls under bsl conditions [17]. These findings suggest tht circulting levels of diponectin nd expression of ADIPOR genes ply n importnt role in the regultion of skeletl muscle insulin ction. It is uncler whether intrcellulr diponectin signlling might lso be bnorml in muscle from insulin-resistnt individuls. Previous work indictes tht AMP-ctivted protein kinse (AMPK) nd p38 mitogen-ctivted protein kinse (p38mapk) re downstrem of the ADIPOR, but do not interct directly with ADIPORs. This work lso identified the dptor protein contining pleckstrin homology domin, phosphotyrosine binding domin nd leucine zipper motif (APPL1) s protein tht intercts with ADIPOR1, using the intrcellulr N-terminl portion of ADIPOR1 s bit in yest two-hybrid screen [18]. APPL1 is 79 kd protein, which contins severl distinct domins, including pleckstrin homology domin, phosphotyrosine-binding domin nd leucine zipper motif embedded in the coiled-coil region [19]. APPL1 intercts with numerous other receptors, including follicle-stimulting hormone receptor, EGF receptor, ndrogen receptor nd the deleted in colorectl cncer receptor [2 23]. In ddition, APPL1 intercts with components of the insulin signlling pthwy such s v-kt murine thymom virl oncogene homologue 2, phosphoinositide 3-kinse, regultory subunit 1 nd the p11 subunit of phosphtidylinositol 3-kinse [19, 22]. This indictes tht APPL1 my ct s scffold protein. Overbundnce of APPL1 increses ftty cid oxidtion nd glucose metbolism upon diponectin stimultion by incresing the ctivity of AMPK nd p38mapk [18]. Although APPL1 does not directly interct with AMPK, diponectin-stimulted ctivity

3 2124 Dibetologi (211) 54: of AMPK seems to be regulted in prt by APPL1 interction with the ADIPOR [18]. Some [24], but not ll [25], studies suggest tht AMPK protein levels nd mrna expression re not ltered in skeletl muscle of obese or type 2 dibetic ptients. In light of the evidence connecting diponectin signlling nd AMPK ctivity, nd given both the decresed ft oxidtion in insulin-resistnt muscle nd the role of APPL1 in diponectin signlling, we sought to determine whether APPL1 bundnce or expression is ltered in insulin resistnce. To test our hypothesis, we first sought to detect the presence of APPL1 in humn skeletl muscle, using western blot techniques nd mss spectrometry (MS). We then estblished novel pproch for quntifying protein bundnce using MS to detect chnges between len control, obese control nd type 2 dibetic prticipnts. We lso performed mrna nlysis to determine whether chnges in APPL1 nd ADIPOR1 were present between these groups. Finlly, we exmined the effect of weight loss following britric surgery on skeletl muscle ADIPOR1/2, APPL1 nd AMPK phosphoryltion in obese insulinresistnt individuls. Methods Prticipnts for protein nlysis We recruited 29 prticipnts for protein bundnce nd quntifiction studies. These included len control prticipnts, obese control prticipnts nd type 2 dibetic prticipnts (Tble 1). All studies were pproved by the Institutionl Review Bord of the University of Texs Helth Science Center t Sn Antonio or Arizon Stte University. Results for some prticipnts used in this study hve been published previously [26]. Prticipnts for mrna nlysis Muscle (vstus lterlis) biopsies from 21 prticipnts were used for rel-time quntittive PCR nlysis. Prticipnt ctegories were len control, obese control nd type 2 dibetic (Tble 2). The study protocol ws pproved by the Institutionl Review Bord of University of Texs Helth Science Center t Sn Antonio. All prticipnts gve written informed consent. Effects of weight loss on diponectin signlling We lso studied six (ge 38±4 yers, BMI 48.5±4.8 kg/m 2, HbA 1c 6.±.1% [men±sem]) non-dibetic morbidly obese prticipnts with the metbolic syndrome, who underwent britric surgery (Roux-en-Y gstric bypss) (Tble 3). Vstus lterlis muscle biopsies were obtined during surgery nd 6 months following surgery, nd were used for rel-time quntittive PCR nlysis nd determintion of AMPK phosphoryltion. These studies were pproved by the Bylor College of Medicine Institutionl Review Bord. Orl glucose tolernce test Bseline blood smples for determining plsm glucose, NEFA nd insulin concentrtions were drwn t 3, 15 nd min. At time, prticipnts ingested 75 g glucose in 3 ml wter. Plsm glucose, NEFA nd insulin concentrtions were mesured t 15 min intervls for 2 h. Euglycemic hyperinsulinemic clmp with muscle biopsies A euglycemic hyperinsulinemic clmp ws performed to determine insulin sensitivity nd to obtin humn skeletl muscle biopsy s previously described [27]. Bsl biopsies were used for ll protein bundnce nd MS nlyses. A clmp ws completed to determine prticipnts insulin sensitivity. Anlyticl determintions Plsm glucose ws mesured using the glucose oxidse method (Beckmn Instruments, Fullerton, CA, USA). Plsm insulin concentrtions were mesured by rdioimmunossy (Dignostic Products, Los Angeles, CA, USA). Plsm diponectin concentrtion ws mesured by ELISA (Linco Reserch, St Chrles, MO, USA) Muscle biopsy processing Frozen muscle biopsies were homogenised in detergent contining lysis buffer s described [28]. Briefly, muscle biopsies were homogenised Tble 1 Chrcteristics of prticipnts used for protein nlysis Vlues re given s men±sem, unless indicted otherwise *p<.5 nd ***p<.1 vs len control; p<.5 vs len nd obese control; p<.5 vs len nd obese control Chrcteristic Len control Obese control Type 2 dibetes Age (yers) 36±3 41±3 48±2* Women (n) Men (n) BMI (kg/m 2 ) 25.8± ± ±2.4 Fsting glucose (mmol/l) 5.5±.2 5.4±.1 8.9±1.3 Len prticipnts (%) 76.2± ± ±3.3* HbA 1c (%) 4.8±.1 5.±.1 7.6±.8 M vlue (mg kg 1 min 1 ) 6.7±.6 4.1±.4 1.9±.7***

4 Dibetologi (211) 54: Tble 2 Chrcteristics of prticipnts used for mrna nlysis Vlues re given s men±sem, unless indicted otherwise *p<.5 nd ***p<.1 vs len control; p<.5 vs len nd obese control Chrcteristic Len control Obese control Type 2 dibetes Age (yers) 32±3 34±3 5±4 Women (n) Men (n) BMI (kg/m 2 ) 24.2± ± ±3.3* Fsting glucose (mmol/l) 5.1±.2 5.2±.1 7.2±.7 Len prticipnts (%) 81.6± ± ±3.1 HbA 1c (%) 4.7±.1 5.1±.1 6.9±.4 M vlue (mg kg 1 min 1 ) 6.3±.5 4.1±.6* 2.7±.5*** while still frozen in buffer (1 μl/mg tissue) consisting of (finl concentrtions): 5 mmol/l HEPES, ph 7.6, 15 mmol/l NCl, 2 mmol/l sodium pyrophosphte, 2 mmol/l β-glycerophosphte, 1 mmol/l NF, 2 mmol/l sodium orthovndte, 2 mmol/l EDTA, ph 8., 1% (vol./vol.) IGEPAL, 1% (vol./vol.) glycerol, 2 mmol/l phenylmethylsulfonyl fluoride, 1 mmol/l MgCl 2, 1 mmol/l CCl, 1 μg/ml leupeptin nd 1 μg/ml protinin. Biopsies were homogenised with polytron homogeniser for 2 s, cooled on ice for 3 min nd then centrifuged for 2 min t 1, g nd 4 C. Biopsy superntnt frctions were frozen until use. Protein concentrtions were determined using the Lowry method [29]. Immunoprecipittion, gel electrophoresis nd western blot nlysis APPL1 ws immunoprecipitted using n nti- APPL1 ntibody (gift from L. Q. Dong). Protein A sephrose beds (Sigm, St Louis, MO, USA) were rehydrted for 2 h t 4 C in 1 PBS (Gibco, Grnd Islnd, NY, USA). Beds were distributed eqully between smples, wshed three times in PBS nd then incubted for 1 h t room temperture in 3 μl PBS with nti-appl1 ntibody (2 μl). Beds were gin wshed three times with PBS nd spirted till dry. Muscle lystes (2.5 mg) were dded to beds nd incubted overnight t 4 C. After incubtion, lystes were removed nd the beds wshed Tble 3 Metbolic nd nthropometric vribles before nd fter weight loss subsequent to britric surgery Vrible Before weight loss After weight lost Body weight (kg) 135.2± ±1.1** BMI (kg/m 2 ) 48.5± ±2.9** Fsting insulin (pmol/l) 161.8± ±6.3** Fsting glucose (mmol/l) 5.8±.3 4.7±.1** HOMA-IR 6.±.5 2.6±.2** Adiponectin (μg/ml) 6.5± ±.9** Vlues re given s men±sem **p<.1 for difference between before vs fter weight loss HOMA-IR, HOMA of insulin resistnce three times with PBS, fter which 35 μl 2 SDS ws dded to beds nd the beds boiled t 95 C for 4 min. Protein (immunoprecipitted or whole muscle lyste) ws seprted on 1% SDS-polycrylmide gel, trnsferred onto nitrocellulose membrne nd blocked for 1 h in 1% (wt/ vol.) BSA t room temperture. Membrnes were incubted for 1 h t room temperture with nti-appl1 or ntiβ-ctin (Cell Signling Technology, Dnvers, MA, USA) primry ntibodies. Membrnes were wshed three times nd incubted for 1 h t room temperture with secondry ntibodies (nti-rbbit, 1:15; GE Helthcre, Pisctwy, NJ, USA). Membrnes were then wshed nd proteins visulised using n electrochemoluminscence rection (Perkin Elmer, Boston, MA, USA). The densities of the bnds corresponding to the proteins of interest were mesured using softwre pckge (Quntity One, Versdoc; Bio-Rd, Hercules, CA, USA). The rtio of the densities of APPL1 to β-ctin ws nlysed by one-wy ANOVA. Significnce ws defined s p<.5. Antibodies The APPL1 ntibody ws rised in rbbits s previously described [18]. Briefly, ntiserum to APPL1 ws rised in rbbits using glutthione-s-trnferse-appl1c. An ntibody to β-ctin ws purchsed from Cell Signling. Electrophoresis, stining nd in-gel digestion Proteins were seprted on 1% SDS-polycrylmide gel contining 1 μg of muscle lyste per prticipnt. Proteins were visulised with Coomssie blue stining. The gel portions contining APPL1 (8 9 kd) nd ctin (37 5 kd) were excised nd prepred s previously described [28, 3], nd s outlined in the electronic supplementry mteril (ESM, Preprtion of smples). Mss spectrometry HPLC-electrospry ionistion (ESI)- MS ws performed on hybrid liner ion trp Fourier trnsform ion cyclotron resonnce mss spectrometer (LTQ FT; Thermo Fisher, Sn Jose, CA, USA), which ws fitted with PicoView nnospry source (New Objective, Woburn, MA, USA). The mss spectrometer ws clibrted weekly ccording to mnufcturer s instructions, chieving

5 2126 Dibetologi (211) 54: mss ccurcies of the clibrnts within 2 ppm. Online cpillry HPLC ws performed using device (Prdigm MS4 micro HPLC; Michrom BioResources, Auburn, CA, USA) with PicoFrit column (New Objective) (75 μmol/l i.d.; pcked with ProteoPep II C18 mteril, 3 nm), mobile phse liner grdient of 2 to 27% (vol./vol.) cetonitrile in.1% (vol./vol.) formic cid in 45 min, hold of 5 min t 27% cetonitrile, followed by step to 5% cetonitrile, hold of 5 min nd then step to 8% (flow rte 25 nl/min). A top ten pproch ws used to identify APPL1 in muscle homogente, wheres trgeted pproch ws used for pek re nlysis; both of these methods hve been described nd vlidted previously by our lb [28, 31]. The top ten pproch ws used for immunoprecipitted APPL1 from humn skeletl muscle nd to verify the ccurcy of the ntibody. All further experiments for MS nlysis were performed on whole-muscle homogentes. Pek re nlysis ws used to determine differences in protein levels between the three prticipnt groups studied. To vlidte western blot dt nd mrna dt, β-ctin ws used s normlistion protein for APPL1 levels; peptides trgeted re listed in ESM Tble 1. We reconstructed ion chromtogrms of the frgment ions of seven peptides for APPL1 nd β-ctin quntifiction. The mss:chrge rtio (m/z) vlues of the seven representtive peptides were plced on the trget list, bsed on ion intensity. The frgment ion m/z vlues hd to be higher thn the precursor ion in order to reduce interference from the bckground or other co-eluting peptide ions. We ssessed the linerity of the β-ctin to APPL1 normlistion quntifiction; complete methods for this cn be found in the ESM (Vlidtion of the use of the APPL1). This method demonstrtes tht our choice of β-ctin s n internl normlistion peptide is vlid for protein quntifiction using the pek re technique. Once these dt were generted, 1 μg of whole-muscle homogente ws run on gel, nd bnds for APPL1 nd β-ctin were excised, trypsinised, deslted nd nlysed by MS s described bove. The rtios of APPL1:β-ctin were used for our quntifiction nlysis. Rel-time quntittive RT-PCR Totl RNA ws isolted using STAT-6 (Tel-Test, Friendswood, TX, USA) nd purified with n mrna kit (Qigen, Vlenci, CA, USA) ccording to the mnufcturer s instructions. Concentrtion ws determined by spectrophotometer t 26/28 nm. Quntittive rel-time PCR ws used to quntify mrna. The specific primers used for quntifiction re listed in ESM Tble 2. mrna fold chnges were clculted reltive to the verge len control vlue quntified reltive to β-ctin. A device (ICycler; Bio-Rd) ws used to quntify mrna levels nd fold chnges were expressed using the 2 ΔΔC t method [32]. AMPK phosphoryltion For western blotting, muscle lystes were prepred s previously described. Proteins (5 μg) from muscle lystes were seprted by 1% SDS- PAGE nd trnsferred to nitrocellulose membrnes. After blocking the membrnes with 5% (wt/vol.) BSA/TBST, membrnes were probed overnight t 4 C with ntibodies ginst nti-phospho-ampk (Thr172) (1:1,; Cell Signling). AMPK phosphoryltion ws normlised for totl AMPK α protein content using pn-α ntibody (1:1; Upstte Biotechnology, Lke Plcid, NY, USA). Bound ntibodies were detected using nti-rbbit immunoglobulin horserdish-peroxidse-linked ntibody nd electrochemoluminscence regents (Perkin Elmer), nd the bnds were quntified using ImgeQunt (GE Helthcre, Pisctwy, NJ, USA) softwre. Sttisticl nlysis Chnges in APPL1 using western blot nd MS were nlysed by ANOVA. Chnges in mrna expression re expressed s fold chnge reltive to len control verge nd sttisticl significnce compred with len control ssessed by ANOVA. Chnges fter weight loss in obese prticipnts were compred using pired t tests. All dt re presented s mens±sem. A vlue of p<.5 ws considered sttisticlly significnt. Results APPL1 protein bundnce determined by western blotting The detection of APPL1 in humn skeletl muscle by immunoprecipittion indictes tht mesurble quntities of the protein were vilble for nlysis (ESM Fig. 1). Whole-muscle homogentes (not immunoprecipitted) were seprted by SDS-PAGE nd western blots nlysed for densitometry. APPL1 nd ctin ntibodies showed specific bnds for the corresponding moleculr weights of APPL1 nd β-ctin. APPL1 protein levels were significntly incresed in type 2 dibetic prticipnts (2.42±.25 rbitrry units [mens±sem]) s compred with len control (1.78±.18) nd obese control prticipnts (1.74±.12; p<.5 for ll) (Fig. 1, b). Our ttempts to co-immunoprecipte endogenous ADIPORs from humn skeletl muscle were unsuccessful. Undetectbility of coimmunoprecipittion my only men tht the interction ws too wek to be mintined during the immunoprecipittion procedure. Although previous investigtors hve reported co-immunopreciption of APPL1 nd ADIPORs, this ws done in cell-bsed system [18]. This is not uncommon, s strong co-immunoprecipittion of the insulin receptor nd IRS1 in yest two-hybrid system hs lso been reported, but this interction is very difficult to observe in humn skeletl muscle (L. J. Mndrino, unpublished results) [33].

6 Dibetologi (211) 54: b APPL1:β-ctin rtio c APPL1:β-ctin rtio Len Obese Type 2 dibetes APPL1 quntifiction using pek re nlysis Vlidtion of APPL1 quntifiction using MS is shown in ESM Fig. 2, with complete methods for APPL1 identifiction described in the ESM (APPL1 identifiction). To confirm chnges of APPL1 levels s visulised by western blots, HPLC-ESI- MS nlysis of muscle homogentes ws performed. A trgeted pproch using peptide m/z vlues for ech APPL1 nd β-ctin peptide ws performed. Seven unique peptides for APPL1 or β-ctin were trgeted. Those identified were quntified ±95% CI. ESM Fig. 3 shows one of seven peptides used for APPL1 quntifiction s n exmple. The pek re is clculted for the unique spectrum identified s the prticulr peptide trgeted. Pek re ws obtined for β-ctin nd APPL1 peptides, nd the rtio of APPL1:β-ctin ws used to numericlly evlute ** Len Obese Type 2 dibetes Len Obese Type 2 dibetes APPL1 β-actin Fig. 1 Humn skeletl muscle APPL1 protein level chnges. Vstus lterlis muscle biopsies were homogenised, seprted by SDS-PAGE nd proteins trnsferred electrophorecticlly to nitrocellulose membrne. Membrnes were exposed to nti-appl1 or β-ctin ntibody nd visulised by chemiluminescence. Density of ech bnd ws determined using Versdoc softwre. b Protein quntifiction, expressed s rtio of APPL1:β-ctin, showing tht APPL1 levels incresed significntly in type 2 dibetic prticipnts s compred with len control nd obese control prticipnts. Vlues men ±SEM (error brs). c Whole skeletl muscle homogentes were used for pek re nlysis. Biopsies were homogenised, seprted by SDS-PAGE nd stined with Coomssie blue. Bnds were cut for APPL1 nd β-ctin, subjected to trypsin digestion nd quntified by MS s described. Protein levels re expressed s rtio of APPL1: β-ctin ( 1 2 ). Vlues re men±sem (error brs); n=8 or 9 per group. *p<.5 * * protein mount in the smple. Pek re nlysis of APPL1 protein showed significntly higher levels in obese control nd type 2 dibetic prticipnts thn in len control prticipnts (Fig. 1c). Identifiction of phosphoryltion of APPL1 t Ser 41 in humn muscle We hd previously identified Ser 43 nd Ser 41 s APPL1 phosphoryltion sites in cultured myocytes (L. Q. Dong, unpublished dt). In ddition, previous reports hve identified Ser 41 s phosphoryltion site on APPL1 in HeL nucler extrcts [34]. Since phosphoryltion ws undetectble using the phospho-specific ntibodies developed for tht study, we used MS to ssess phosphoryltion of APPL1. We trgeted these nd other potentil phosphoryltion sites s predicted by Phosphosite (www. phosphosite.org; ccessed 13 April 211), clculted s loss of 8 D or 98 D on the bsis of 2+ or 3+ chrge stte, respectively. Due to the low levels of APPL1 phosphorylted peptides in whole-muscle homogentes, immunoprecipittion of APPL1 ws performed. Muscle homogente (2.5 mg) ws immunoprecipitted with the APPL1 ntibody, nd phospho-ser 41 trgeted nd identified on the bsis of the 3+ chrge stte. The tndem mss spectrum of pser 41 peptide is shown in Fig. 2. Chnges in APPL1 nd ADIPOR1 mrna expression between prticipnt groups To determine whether chnges in mrna expression of APPL1 nd ADIPOR1 were present in len, obese nd dibetic prticipnts, rel-time PCR ws b Reltive bundnce b V N Q S A L E A V T P S P S F Q Q R happl y y y y y b , 1,1 1,2 1,3 1,4 1,5 m/z y b b14(c 13 ) b y y Fig. 2 Identifiction of Ser 41 s phosphoryltion site. APPL1 phosphoryltion t Ser 41 ws detected in tryptic digests of APPL1 isolted from humn muscle biopsy. APPL VNQSA- LEAVTPpSPSFQQR (pser 41 ) peptide ws nlysed (b) by tndem mss spectrum using HPLC-ESI-MS/MS/MS. The presence of y-series ions contining phosphte strting with y5 support phosphoryltion of Ser 41

7 2128 Dibetologi (211) 54: used. Results indicte similr trend to tht seen in protein levels of APPL1. Obese controls nd type 2 dibetic ptients hd significntly incresed mrna expression of APPL1 compred with len controls (Fig. 3). Skeletl muscle ADIPOR1 expression incresed pproximtely threefold (p<.5) in obese prticipnts compred with len controls, lthough type 2 dibetic ptients did not differ from len control individuls (Fig. 3b). Chnges in skeletl muscle diponectin signlling fter weight loss At 6 months following Roux-en-Y gstric bypss surgery [35], body weight (p<.1), men fsting plsm glucose (p<.1), plsm insulin (p<.1) nd insulin resistnce (HOMA of insulin resistnce) (p<.1) decresed significntly (Tble 3). The improvement in insulin sensitivity ws ssocited with n increse in plsm diponectin levels (6.5±.6 to 11.6±.9 μg/ml, p<.1). Skeletl muscle ADIPOR1 nd ADIPOR2 expression incresed pproximtely twofold (p<.5), while APPL1 expression decresed by pproximtely 5% (p<.5) following weight loss (Fig. 4). Skeletl muscle AMPK phosphoryltion (P-AMPK: AMPK rtio) incresed significntly following weight loss (.47±.5 to.78±.13, p<.5) (Fig. 5). Discussion APPL1 mrna fold chnge b ADIPOR1 mrna fold chnge Len Len * Obese * Obese * Type 2 dibetes Type 2 dibetes Fig. 3 Humn skeletl muscle expression of () APPL1 nd (b) ADIPOR1. Quntittive rel-time PCR ws used to quntify mrna. mrna fold chnges were clculted reltive to verge len control vlue quntified reltive to β-ctin. mrna expression nd fold chnges re expressed using the 2 ΔΔCt method [32]. *p<.5 vs len mrna fold chnge Adiponectin signlling is n intriguing re of reserch due to its nti-inflmmtory, glucose-lowering nd insulinsensitising properties [1 3, 36 38]. Although some components of the pthwy hve been elucidted, mny questions regrding the regultion nd interction of proteins involved remin unnswered. Previous findings hve shown tht diponectin intercts with the C-terminl extrcellulr portion of the ADIPOR [13] nd, upon binding, recruits APPL1 to the N-terminl intrcellulr portion [18]. This binding my fcilitte the downstrem effects of diponectin, which include AMPK nd p38mapk ctivtion, thus leding to incresed ftty cid oxidtion nd glucose uptke [18, 37]. P-AMPK:AMPK rtio 1..5 ADIPOR1 ADIPOR2 APPL1 Fig. 4 Effect of weight loss following britric surgery on ADIPOR1, ADIPOR2 nd APPL1 expression. Quntittive rel-time PCR ws used to quntify mrna. mrna fold chnges were clculted reltive to verge pre-surgery vlue quntified reltive to 18S vlues. mrna expression nd fold chnges re expressed using the 2 ΔΔCt method [32]. *p<.5 Pre Post P-AMPK Totl AMPK Fig. 5 Skeletl muscle AMPK expressed s phosphorylted AMPK (P-AMPK):totl AMPK rtio in six morbidly obese ptients before (pre) nd fter (post) weight loss following britric surgery. *p<.5

8 Dibetologi (211) 54: Previous reports hve indicted tht APPL1 plys n importnt role in the diponectin signlling pthwy by trnsmitting signls from the receptor to downstrem trgets [18, 19]. Although APPL1 hs been identified nd chrcterised in cells, to dte no studies hve identified its presence in humn skeletl muscle. Here we hve identified APPL1 in biopsies tken from humn vstus lterlis muscle nd quntified APPL1 protein nd mrna in smples from len, obese nd type 2 dibetic prticipnts. To determine whether APPL1 ws present in humn skeletl muscle, immunoprecipitted nd superntnt frctions of muscle homogentes were run on western blots, reveling bnd specific for APPL1. To verify tht this bnd ws indeed APPL1, we rn identicl gels, using one for western blot nd the other for Coomssie stining. The Coomssie stin showed very light bnd t ~8 kd, similr to the size of APPL1 (82 kd). We cut this bnd nd performed top ten pproch using the MASCOT (version 2.2; Mtrix Science, London UK) serch engine, which detects the top ten most bundnt proteins in smple. MASCOT detected APPL1 in the muscle homogente smple with sequence coverge of 56%. We lso tested whether APPL2 ws present in the smple, but did not find this protein (dt not shown). Upon verifiction tht the western blot bnd corresponded to APPL1, we rn whole-muscle homogentes to quntify western blots using the APPL1 ntibody. Western blot quntifiction showed APPL1 levels to be significntly higher in type 2 dibetic ptients thn in len nd obese controls. Although western blot cn be useful for quntifying protein levels, we vlidted our APPL1 quntifiction results using MS. In fct, in the present study, we implemented unique MS quntifiction technique using β-ctin peptides s normlistion fctor. To verify the vlidity of this method for quntifiction purposes, incresing mounts of muscle homogente smples were nlysed (5 μg, 75 μg nd 1 μg). Peptides for β-ctin nd APPL1 were trgeted nd Scffold (version 2 6; Proteome Softwre, Portlnd, OR, USA) ws used to quntify pek res. The pek res of the peptides for ech protein were verged nd plotted for regression nlysis. The verges of the peptides were quntified so tht the rtio of APPL1:β-ctin levels represented protein present in the smple. The CV from these rtios ws found to be 6.59%, indicting tht incresing mounts of smple were consistent bsed on the APPL1:β-ctin rtio. For this study, we quntified proteins bsed on pek re s opposed to ion intensity, becuse pek re is the verge pek of mny scns, s opposed to using just one scn for ion intensity, nd therefore reduces vribility. After determining tht β-ctin could be used s normlistion fctor, we then quntified APPL1 levels in humn skeletl muscle smples using pek re nlysis. The pek re rtio showed significntly higher APPL1 levels in obese control nd type 2 dibetic prticipnts compred with len control. The difference in dt between western blot nd MS could be explined by the vribility in immunoblot nlysis. Due to the mrna nd MS dt, nd lso bsed on linerity vlidtion, we believe tht MS nlysis of protein bundnce is more likely to be correct. This indictes tht APPL1 levels re higher in n insulin-resistnt stte (obese controls nd type 2 dibetic ptients) nd tht the body my be compensting for decresed levels of diponectin, nd therefore trying to increse signlling downstrem of the ADIPOR through incresed APPL1 production. We lso used quntittive rel-time PCR to determine chnges in APPL1 mrna mong the three experimentl groups. APPL1 mrna incresed significntly in obese control nd type 2 dibetic prticipnts, s compred with len control. This verifies our protein dt, showing tht in stte of insulin resistnce, APPL1 mrna expressison is incresed, possibly representing compenstory mechnism in skeletl muscle to enhnce diponectin signlling. Additionlly, ADIPOR1 mrna expression ws significntly incresed in obese compred with len controls, but ws not ltered in type 2 dibetic ptients. The increse in ADIPOR1 mrna in obese controls my be n dditionl compenstory mechnism triggered by the decresed diponectin levels in obese nd type 2 dibetic ptients. ADIPOR1 mrna expression in type 2 dibetic ptients my become exhusted, thus leding to levels similr to those in len controls. The combintion of compenstory mechnisms for APPL1 nd ADIPOR1 due to decresed diponectin levels in the obese stte illustrtes how this signlling pthwy is ltered in insulin resistnce (Fig. 6). To further understnd how APPL1 nd ADIPORs chnge in reltion to insulin resistnce, skeletl muscle ADIPOR1/2 nd APPL1 expression ws determined following weight loss fter britric surgery in morbidly obese, non-dibetic prticipnts with the metbolic syndrome. Following weight loss, plsm diponectin incresed significntly nd ws ssocited with twofold Obesity Dibetes Post weight loss Adiponectin levels c ADIPOR1/R2 b c APPL1 Fig. 6 Schemtic representtion of protein or mrna chnges in diponectin, ADIPOR1/2 nd APPL1 ssocited with obesity, dibetes nd post weight loss. Compenstory mechnism compred with len; b exhusted system compred with len; c normlised system compred with pre-weight loss c

9 213 Dibetologi (211) 54: increse in both ADIPOR1 nd ADIPOR2 expression. APPL1 expression ws significntly reduced nd skeletl muscle AMPK phosphoryltion incresed significntly, suggesting tht the correction of hyperglycemi nd hypodiponectinemi following weight loss is ssocited with reduction in APPL1 expression. This normlistion of APPL1 nd ADIPOR1/R2 levels fter weight loss illustrtes the compenstory mechnism to chnges in diponectin levels pre nd post-surgery (Fig. 6). In ddition to mrna nd protein level chnges, we lso hve identified Ser 41 s phosphoryltion site in APPL1 in humn skeletl muscle. This phosphoryltion site hs previously been identified in HeL cell nucler extrct, lthough this is the first time it hs been reported in humn skeletl muscle [34]. Currently, we re testing the kinse responsible for this phosphoryltion site, nd whether this site plys functionl role in regulting APPL1 function in diponectin signlling. In conclusion, APPL1 levels re ssocited with insulin resistnce, during which the body my be ttempting to compenste for the decresed diponectin levels found in this stte. In ddition, APPL1 is phosphorylted in humn skeletl muscle t Ser 41, mechnism of functionl regultion tht is now under investigtion nd my shed new light on the diponectin signlling pthwy. Improvements in hyperglycemi nd hypodiponectinemi following weight loss re ssocited with enhnced skeletl muscle diponectin signlling, s well s AMPK phosphoryltion nd reduction in APPL1 levels. Acknowledgements This work ws supported in prt by grnts from the Americn Dibetes Assocition 1-6-CR-3 (to M. Bjj) nd the Ron McDonld Foundtion t St Luke s Episcopl Hospitl 8RDM8 (to M. Bjj), s well s by Mrilyn Fishmn Grnt for Dibetes Reserch from the Endocrine Fellows Foundtion (to S. Shhni) nd grnts NIH R1DK8175-1A1 (to Z. Yi) nd NIH R1DK66483 (to L. J. Mndrino). 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