ARTICLE. Keywords Insulin resistance. NO synthase. Obesity. Skeletal muscle. Type 2 diabetes

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1 Diabetologia (214) 57: DOI 1.7/s ARTICLE Counterating neuronal nitri oxide synthase proteasomal degradation improves gluose transport in ulin-resistant skeletal musle from Zuker fa/fa rats Karima Mezghenna & Jérémy Leroy & Jaqueline Azay-Milhau & Didier Toush & Françoise Castex & Sylvain Gervais & Viviana Delgado-Betanourt & René Gross & Anne-Dominique Lajoix Reeived: 11 Marh 213 /Aepted: 3 September 213 /Published online: 2 November 213 # Springer-Verlag Berlin Heidelberg 213 Eletroni supplementary material The online version of this artile (doi:1.7/s ) onta peer-reviewed but unedited supplementary material, whih is available to authorised users. K. Mezghenna: J. Leroy : J. Azay-Milhau : D. Toush : F. Castex : S. Gervais: V. Delgado-Betanourt : R. Gross : A.<D. Lajoix () Centre for Pharmaology and Innovation in Diabetes, University Montpellier 1, EA 7288, 15 Avenue Charles Flahault, BP 14491, 3493 Montpellier edex 5, Frane anne-dominique.lajoix@univ-montp1.fr Abstrat Aims/hypothesis Insulin-mediated gluose transport and utilisation are dereased in skeletal musle from type 2 diabeti and gluose-intolerant individuals beause of alterations in ulin reeptor signalling, GLUT4 transloation to the plasma membrane and mirovasular blood flow. Catalyti ativity of the musle-speifi isoform of neuronal nitri oxide synthase () also partiipates in the regulation of gluose transport and appears to be dereased in a relevant animal model of drasti ulin resistane, the obese Zuker fa/fa rat. Our objetive was to determine the moleular mehanisms involved in this defet. Methods Isolated rat musles and primary ultures of myoytes were used for western blot analysis of protein expression, immunohistohemistry, gluose uptake measurements and GLUT4 transloation assays. Results expression was redued in skeletal musle from fa/fa rats. This was aused by inreased ubiquitination of the enzyme and subsequent degradation by the ubiquitin proteasome pathway. The degradation ourred through a greater interation of with the haperone heat-shok protein 7 and the o-haperone, arboxyl terminus of Hs7- interating protein (CHIP). In addition, an alteration in sarolemmal loalisation was observed. We onfirmed the impliation of breakdown in defetive ulinindued gluose transport by demonstrating that blokade of proteasomal degradation or overexpression of improved basal and/or ulin-stimulated gluose uptake and GLUT4 transloation in primary ultures of ulin-resistant myoytes. Conlusions/interpretation Reovery of in ulinresistant musles should be onsidered a potential new approah to address ulin resistane. Keywords Insulin resistane. NO synthase. Obesity. Skeletal musle. Type 2 diabetes Abbreviations CHIP Carboxyl terminus of Hs7-interating protein enos Endothelial nitri oxide synthase Hsp Heat-shok protein Neuronal nitri oxide synthase NO Nitri oxide NOS Nitri oxide synthase PIN Protein inhibitor of neuronal nitri oxide synthase SNP Sodium nitroprusside ZDF Zuker diabeti fatty Introdution Nitri oxide (NO) is a short-lived gas produed by a family of enzymes alled NO synthases (NOSs). Three isoforms have been identified, inluding onstitutive neuronal (n)nos, endothelial (e)nos and ytokine-induible NOS (inos) [1]. Human and rodent skeletal musle fibres mainly express [2], whereas enos, whih is also present in some musle fibres in rodents [3], is predominantly found in the endothelium of blood vessels within human skeletal musle

2 178 Diabetologia (214) 57: [2]. A unique isoform, μ, a splie variant of α that displays an ertion of 34 amino aids between exons 16 and 17 without any modifiation of its atalyti ativity, is expressed in skeletal musle [4]. is mainly but not exlusively distributed along the sarolemma of positive fibres [2], as a omponent of the dystrophin glyoprotein omplex [5]. is involved in the regulation of ontratile fore, ulin- and exerise-stimulated gluose transport, energy metabolism and blood flow (for review, see [6] and [7]). Interestingly, the signalling pathway is altered in various pathologies inluding Duhenne musular dystrophy and type 2 diabetes. Indeed, type 2 diabetes is haraterised by both panreati beta ell seretory defet and ulin resistane of the peripheral tissues and liver. Skeletal musle displays dereased ulin-mediated gluose transport and utilisation in type 2 diabeti patients, as well as in those with gluose intolerane [8], as a onsequene of impaired ulin signalling and GLUT-4 transloation pathways (for review, see [9]). The demonstration of a role for in ulin resistane emerged from a study by Shankar et al [1], whih showed that -knokout mie were ulin resistant at the level of peripheral tissues. In addition, Young and Leighton provided evidene for the possible involvement of dereased NOS atalyti ativity in abnormal gluose transport and utilisation in the ulin-resistant Zuker fa/fa rat [11]. The aim of this study is to investigate the moleular mehanisms involved in the alteration of NOS atalyti ativity observed in the Zuker fa/fa rat. Zuker fa/fa rats display a mutation in the leptin reeptor [12], with hyperphagia, obesity, ulin resistane and hyperulinaemia as phenotypi harateristis. We found that expression was redued in the skeletal musle of fa/fa rats beause of inreased degradation of the enzyme by the ubiquitin proteasome pathway. Importantly, restoration of expression and atalyti ativity improved gluose uptake and GLUT4 transloation in ulin-resistant myoytes. Methods Animals Male Zuker fa/fa rats and their ontrol littermates, fa/+ rats, were purhased from Harlan (Blakthorn, UK) and Zuker diabeti fatty (ZDF) rats were purhased from Charles River (L Arbresle, Frane) (see Eletroni Supplementary Material [ESM] Methods). Institutional guidelines for animal are were followed and the protool was approved by our University ethial ommittee. Zuker fa/fa rats were killed at the age of 9 11 weeks and ZDF rats were killed at the age of 1 weeks. Real-time reverse transription (RT)-PCR Total RNA from gastronemius musles was extrated with RNA Now reagent (Biogentex, League City, TX, USA). First-strand DNA was obtained from 2 μg total RNA using SuperSript II RNAse H Reverse Transriptase (Invitrogen, Paisley, UK). Real-time quantitative PCR was arried out to assess the gene expression of nnos (otherwise known as Nos1), protein inhibitor of (Pin; otherwise known as Dynll1) and Gadph (as an invariant housekeeping gene) using speifi primers. PCR was performed with FastStart DNA Master SYBR Green I mix (Rohe, Basel, Switzerland) on a LightCyler (Rohe). Western blotting and immunopreipitation Setions from the middle of the musle were homogenised in lysis buffer ontaining 1% Triton X-,.1% SDS and protease inhibitors (1 ml per mg tissue; Rohe) using a Polytron homogeniser (IKA, Stanfen, Germany). Prote (75 μg) were separated on a 7.5% SDSpolyarylamide gel for and on a 14% triine polyarylamide gel for PIN. Inubation with primary antibodies (see ESM Methods) was performed overnight. Signals were aquired with a Vilber Lourmat imager (Marne-la-Vallée, Frane) and quantified by GeneTools image analysis software (Syngene, Cambridge, UK). In relative quantifiation graphs, protein levels are expressed relative to α-tubulin. Eah experiment shown is representative of three independent western blots. For subellular frationation, skeletal musles were homogenised in buffer ontaining 2 mmol/l Tris (ph 7.4), 15 mmol/l NaCl and protease inhibitors. The supernatant frations were entrifuged at 5, g and the membrane pellets were solubilised for 1 h at 4 C in lysis buffer ontaining 1% Triton X-,.5% Nonidet P-4 and.5% sodium deoxyholate. For immunopreipitation experiments, musles were homogenised in lysis buffer ontaining 1% Triton X-,.5% Nonidet P-4, protease inhibitors and N-ethylmaleimide (Sigma-Aldrih, Steinheim, Germany). or arboxyl terminus of Hs7-interating protein (CHIP) was immunopreipitated from a 2 mg extrat with polylonal anti- (BD Biosienes, Franklin Lakes, NJ, USA) or polylonal anti-chip (Santa Cruz Biotehnologies, Santa Cruz, CA, USA) antibody overnight and prote were immunoblotted as desribed above. Immunofluoresene Setions from gastronemius musles (8 μm thik) were fixed with 2% paraformaldehyde and immunostained overnight with anti- (EuroProxima, Arnhem, the Netherlands), anti-pin (Santa Cruz) or anti-α1 syntrophin (Sigma-Aldrih) antibodies. Fluoresene was observed with the Zeiss (Oberkohen, Germany) LSM 78 onfoal mirosope using the Montpellier RIO Imaging platform (Montpellier, Frane). A negative ontrol was performed by inubating setions with the seondary antibody.

3 Diabetologia (214) 57: Inubation experiments with isolated musles Eah freshly isolated musle was fixed on a plasti holder at its original length and plaed vertially in an assay tube. After a 15 min preinubation period in Krebs Henseleit biarbonate buffer (ph 7.4), musles were inubated in the presene or absene of 5 μmol/l MG 132 (Sigma-Aldrih) for 2 h at 37 C, with regular 95% O 2 /5% CO 2 bubbling. Isolation and ulture of satellite ells Satellite ells were isolated from rat gastronemius musles, as desribed in ESM Methods [13, 14]. For differentiation, myoblasts were seeded on 12-well oated plates in growth medium. After 5 days, the ells were inubated with DMEM ontaining 2% horse serum (differentiation medium) for another 5 day period. Treatments of satellite ells Musle ells were first differentiated for 3 days. For overexpression, an expression vetor ontaining nnosα DNA (pcr3.1, Invitrogen) was introdued into musle ells using PolyJet reagent (SignaGen, Ijamsville, MA, USA). Musle ells werealsotreatedfor48hwith2μmol/l ell-permeant ubiquitin proteasome system inhibitory Tat peptide, GRKKRRQRRRGGKAVDLSHQPS (EZBiolab, Carmel, IN, USA), or Tat alone, in ontrol onditions [15]. expression was evaluated by western blot 48 h after eah treatment and musle ells were subjeted to gluose uptake or GLUT4 transloation assay. Gluose uptake in primary myoytes The day of the experiment, ells were first starved and then inubated for 1 h in KRB buffer, ontaining the ompounds listed in the ESM Methods. [1,2-3 H]-2-deoxygluose uptake was performed for 5 min (see ESM Methods). Radioativity was normalised to the total protein onentration (pm mg 1 min 1 ). Changes are expressed in per ent of basal ondition value (absene of ulin). Transloation of GLUT4 Musle ells were stimulated for 3 min, fixed and inubated overnight with an anti-glut4 antibody (Alpha Diagnosti, San Antonio, TX, USA). Immunoreativity was revealed with a peroxidase-onjugated antibody and a olorimetri reation (see ESM Methods). NOS atalyti ativity assay NOS atalyti ativity was estimated in musle extrats by measuring the prodution of radiolabelled [ 3 H]itrulline from [ 3 H]arginine (MP Biomedials, Irvine, CA, USA) aording to the manufaturer s reommendation (Nitri Oxide Synthase Assay Kit, Merk Millipore, Billeria, MA, USA). NOS ativity was measured after a 1 h reation at 37 C and after separation of labelled itrulline from arginine by affinity hromatography. Statistial analysis Experimental values represent the mean of n experiments and are plotted on graphs as means ± SEM. The data were analysed using a Student s t test or ANOVA where appliable. Results Dereased expression in skeletal musle from Zuker fa/fa rats Young and Leighton [11] previously reported dereased NOS atalyti ativity in skeletal musle from Zuker fa/fa rats. We onfirmed this defet in the ulinresistant gastronemius musle, whih displayed a 4% redution in NOS atalyti ativity (p <.1, n =4) (Fig. 1a). By western blot, we observed a 42% derease in the a NOS atalyti ativity (pm/µg protein) enos e PIN 6 kda enos protein level PIN protein level b protein level d f p- p-/ level Lean ZDF protein level µ splie variants Lean ZDF Obese ZDF Obese ZDF Fig. 1 NOS expression and atalyti ativity in the gastronemius musle from Zuker fa/fa and fa/+ rats.(a) NOS atalyti ativity in musle from Zuker fa/+ vs fa/fa rats (n =4). Expression of (b) (arrows indiate different splie variants of ), () enos,(d) p- on Ser1412 (results are normalised aording to the protein ontent) and (e) PIN by western blot in fa/+ and fa/fa rats. (f) Expression of by western blot in gastronemius musle from lean and obese ZDF rats. Eah western blot is representative of three independent experiments. p <.5; p <.1

4 18 Diabetologia (214) 57: protein ontent in musle from fa/fa vs fa/+ rats (p <.5) (Fig. 1b). Interestingly, splie variants of, inluding β laking the N-terminus PDZ domain [16], ould also be deteted in skeletal musle (Fig. 1b). By ontrast, enos expression was found to be unhanged in fa/fa vs fa/+ rats (Fig. 1) and inos was not expressed in musle from fa/fa rats (data not shown). We then looked at phosphorylation on Ser1412, whih has previously been shown to transiently ativate the enzyme ativity [17], and observed a 2.15-fold inrease in the phospho-/ ratio in musle from fa/fa rats (Fig. 1d). Moreover, has been reported to be modulated by an endogenous protein inhibitor, PIN, that inhibits NO prodution [18]. However, no hange in PIN protein expression ould be observed in musle from fa/fa rats (Fig. 1e). Finally, in gastronemius musles from diabeti ZDF rats, we found, as in Zuker fa/fa rats, a 42% redution in expression in obese vs lean rats, but the proportion of eah variant differed between the two animals (Fig. 1f). Thus, NOS defet is related to dereased expression in ulin-resistant musle, despite a slight inrease in phosphorylation, probably aimed at ompensating its atalyti deline. Changes in subellular loalisation We analysed whether subellular loalisation ould be altered in ulin-resistant musle. As ompared with fa/+ rats (Fig. 2a) a drasti derease in sarolemmal immunoreativity ould be observed in fa/fa rats (Fig. 2b), whih ontrasted with the more slightly redued ytoplasmi staining. Subellular loalisation of PIN (Fig. 2d) oinided with that of in skeletal musle in fa/+ rats (Fig. 2), and the sarolemmal loalisation of PIN was also found to be dereased in fa/fa rats (Fig. 2f), as for (Fig. 2e). In addition, expression (data not shown) and loalisation of α1-syntrophin, known to anhor at the level of the sarolemma [5], were unmodified in musle from fa/fa rats (ompare Fig. 2h, j). Finally, we onfirmed hanges in loalisation by subellular frationation of gastronemius musle. The purity of eah fration was measured by the presene of extraellular-signal-regulated kinase 1/2 (ytoplasmi) and IL-1 reeptor type 1 (partiulate) (Fig. 2k). We found a 17% and 58% derease in ytoplasmi and partiulate, respetively, in musle from fa/fa rats (Fig. 2k). The relative level of in eah fration appeared to be non-omparable beause of the experimental onditions. Evidene for degradation by the ubiquitin proteasome pathway Surprisingly, we did not observe any signifiant variation in nnos mrna in fa/fa rats using real-time RT- (reverse transription) PCR (n =6) (Fig. 3a), suggesting possible degradation by the ubiquitin proteasome system. In the presene of the proteasome inhibitor MG 132 (5 μmol/l), NOS atalyti ativity was found to be unhanged in fa/+ rats, whereas in fa/fa rats, it was restored to a level similar to that observed in ontrol rats (47% inrease, p <.5, n =4) (Fig. 3b). Moreover, MG 132 treatment normalised protein ontent in fa/fa rats (p <.5), Fig. 2 Subellular loalisation of, PIN and α1-syntrophin in gastronemius musle setions from Zuker fa/+ and fa/fa rats analysed by immunofluoresene. Setions from fa/+ (a,, d, g, h)andfa/fa (b, e, f, i, j) rats were labelled for (a, b,, e, g, i), PIN (d, f) and α1-syntrophin (h, j). Sale bars, 5 μm. (k) Expression of by western blot in ytoplasmi and partiulate frations of gastronemius musle from fa/+ and fa/fa rats. The purity of eah fration was heked by evaluating the presene of extraellular-signalregulated kinase 1/2 (ERK1/2; ytoplasmi fration, CF) and IL-1 reeptor type 1 (IL1R1; partiulate fration, PF). p <.5; p <.1 a b g h e d f i k ERK1/2 36 kda Cytoplasmi CF Partiulate j PF CF PF 98 kda CF PF CF PF IL1R1 Cytoplasmi level Partiulate level 3 2 5

5 Diabetologia (214) 57: a b enos protein level enos protein level Quantifiation nnos/gapdh MG 132 MG 132 MG 132 MG 132 NOS ativity (pm/µg protein) whereas enos expression was unhanged by MG 132 (Fig. 3). As enos is also ativated by phosphorylation on serine 1177 by Akt [19], we measured enos phosphorylation by western blot and found no hanges in fa/fa vs fa/+ rats (data not shown). Interestingly, the entire μ was more subjet to proteolyti degradation than the other splie variants laking the PDZ domain. In addition, using immunopreipitation, we observed an aumulation of ubiquitinated protein in musle from fa/fa as ompared with fa/+ rats, whih was reinfored by treatment with MG 132 (Fig. 3d). Finally, we investigated whether the Ca 2+ -dependent protease alpain ould also be involved in degradation, as previously desribed [2]. We found 2% dereased expression of alpain-3 and -1/2 in musle from fa/fa rats (p <.5) (Fig. 3e). Overall, our results suggest that the ubiquitin proteasome system, rather than the protease d IP Ubiquitin ubiquitination e MG 132 MG 132 ubiquitination 98 kda Calpain-3 Calpain-1/2 Calpain protein level Fig. 3 Evidene for degradation by the ubiquitin proteasome pathway in isolated gastronemius musle from Zuker fa/fa vs fa/+ rats. (a) Expression of nnos analysed by real-time RT (reverse transription)-pcr in fa/+ and fa/fa rats (n =6). (b) Effet of the proteasome inhibitor MG 132 (5 μmol/l) on atalyti ativity in fa/+ vs fa/fa rats (n =4). () Effet of MG 132 (5 μmol/l) on and enos expression in fa/+ vs fa/fa rats and relative quantifiation of the band intensities. (d) Ubiquitination of in fa/+ and fa/fa rats in the presene or absene of MG 132. was immunopreipitated and the presene of ubiquitin and revealed by western blot. (e) Expression of alpa by western blot in fa/+ and fa/fa rats. p <.5 alpain, is involved in degradation in ulin-resistant skeletal musle. Effet of proteasomal degradation blokade on gluose uptake in primary musle ells In isolated rat musle, we onfirmed dereased ulin sensitivity in fa/fa vs fa/+ musles ( 21±5%, from 3.2 to 2.4 pm mg 1 min 1 ). For further experiments, we isolated satellite ells from gastronemius musle and differentiated them into myotubes to perform gluose-uptake experiments. Basal gluose uptake was redued by 21% in musle ells from fa/fa vs fa/+ rats (from 3,3±245 to 2,364±223 pm mg 1 min 1 ; p <.5; n =6), as previously desribed [21]. In fa/+ rats, ulin dose-dependently stimulated gluose uptake at 1 nmol/l (+24±2.2%; p <.1; n =6) and nmol/l (+44±6.5%; p <.1; n =6) (Fig. 4a). Insulin slightly promoted gluose a d f g nmol/l nmol/l 1 nmol/l nmol/l 1 nmol/l 1 nmol/l + MG MB + MB + MB + MB nmol/l 1 nmol/l 1 nmol/l + MB b e h nmol/l nmol/l protein level 1 nmol/l nmol/l nmol/l 1 nmol/l 1 nmol/l + MG MG MB MB nmol/l nmol/l1 nmol/l1 nmol/l + MB + MB Fig. 4 Effet of proteasomal degradation blokade on gluose uptake in primary musle ells from Zuker fa/fa vs fa/+ rats. Effet of inreasing onentrations of ulin (;, 1, nmol/l) on gluose uptake in musle ells from fa/+ (a) and fa/fa (b) rats. () Expression of by western blot in primary musle ells from fa/+ and fa/fa rats. Effet of MG 132 (5 μmol/l) on gluose uptake in musle ells from fa/+ (d)andfa/fa (e) rats, with or without 1 nmol/l ulin. (f)effetof Methylene Blue (MB; 1 μmol/l) on expression in isolated musle from fa/+ vs fa/fa rats. Effet of Methylene Blue on gluose uptake in musle ells from fa/+ (g) and (h) fa/fa rats, with or without 1 nmol/l ulin. p <.5; p <.1; p <.1

6 182 Diabetologia (214) 57: uptake at the nmol/l onentration in ulin-resistant musle ells (+9±4.6%; p <.5; n =6) (Fig. 4b), as observed by others in type 2 diabeti patients [22]. We onfirmed that expression remained redued in ultured myoytes, as in entire musles (Fig. 4). The proteasome inhibitor MG 132 (5 μmol/l) did not modify gluose uptake in fa/+ musle ells (n =5) (Fig. 4d), whereas MG 132 inreased gluose uptake by 18±5% (p <.1; n =5) and 3±6% (p <.1; n =5), respetively, in the absene and presene of ulin (1 nmol/l) in fa/fa rats (Fig. 4e). As degradation is dependent upon the heat-shok protein (Hsp)7 haperone mahinery [23], we also used Methylene Blue, an inhibitor of Hsp7-dependent proteasomal degradation [24], and observed that the inhibitor was able to restore expression in musle from fa/fa rats (Fig. 4f). In addition, Methylene Blue (1 μmol/l), whih was ineffetive in fa/+ rats (Fig. 4g), improved basal and ulin-stimulated (1 nmol/l) gluose uptake by 42±6% and 61±11.5%, respetively, in fa/fa musle ells (p <.1; n =5) (Fig. 4h). Finally, several lysine residues have been predited to be potential ubiquitination sites, using the UbPred program ( [25]. As suggested by Fig. 3, the main site is probably loalised in the N-terminus of the enzyme. To speifially ounterat proteasomal degradation, we designed an inhibitory peptide targeting a high onfidene ubiquitination site loated at Lys131 of rat and oupled it to the Tat sequene, to render it ell permeant. Indeed, inhibitory peptides have been shown to speifially ounterat the proteasomal degradation of a target protein [26]. Moreover, ell-penetrating peptides have been shown to be easily aptured in primary ardiomyoytes in vitro [27]. In musles ells from fa/fa rats, the Tat peptide inreased expression by 84% (Fig. 5a) and improved gluose uptake in both basal and ulin-stimulated (1 nmol/l) onditions (+11±3.5%; p <.1; n =7) (Fig. 5b). Thus, we onlude that speifially ounterating proteasomal a Tat Tat peptide protein level Tat Tat peptide b nmol/l + Tat nmol/l 1 nmol/l + Tat + Tat peptide 1 nmol/l + Tat peptide Fig. 5 Effet of restoration by the inhibitory Tat peptide on gluose uptake in primary musle ells from Zuker fa/fa rats. (a) Western blot analysis of expression with ell-permeant inhibitory Tat peptide or Tat alone (2 μmol/l). (b) Effet of Tat peptide on gluose uptake in ells from fa/fa rats, with or without 1 nmol/l ulin (). p <.1 degradation improves gluose uptake in ulin-resistant musle ells. Effet of restoration on gluose uptake in primary musle ells We first heked whether musle ells from fa/fa rats retain their sensitivity to sodium nitroprusside (SNP), as previously desribed [21]. SNP (1 mmol/l) enhaned basal (+29±4%; p <.1; n =5) and ulin-stimulated (1 nmol/l) (+44±6%; p <.1; n =5) gluose uptake in myoytes from fa/+ rats (Fig. 6a). Cells from ulin-resistant fa/fa rats remained sensitive to SNP in both basal (+3±6%; p <.1; n =5) and ulin-stimulated (1 nmol/l) (+48±7%; p <.1; n =5) gluose uptake (Fig. 6b). When musle ells from fa/fa rats were transfeted with an expression vetor oding for α, expression was inreased threefold (Fig. 6) and gluose uptake improved by 17±8% (p <.5; n =5) and 28±9.8% (p <.1; n =5)inbasalandulin- a 15 5 nmol/l nmol/l + SNP protein level e GLUT4 transloation (%) nmol/l d 1 nmol/l + SNP 15 nmol/l nmol/l 1 nmol/l 1 nmol/l ontrol ontrol b 15 5 nmol/l 5 nmol/l nmol/l 1 nmol/l 1 nmol/l ontrol ontrol f 64 kda GLUT4 GLUT4 protein level 15 5 nmol/l 1 nmol/l 1 nmol/l + SNP + SNP Fig. 6 Effet of α restorationbyoverexpressionongluoseuptake and GLUT4 transloation in primary musle ells from Zuker fa/fa rats. Effet of SNP (1 mmol/l) on gluose uptake in fa/+ (a)andfa/fa (b)rats, with or without 1 nmol/l ulin (). () Western blot analysis of overexpression after transfetion with nnosα DNA () or an empty vetor (ontrol). (d) Effetofα overexpression () on gluose uptake in musle ells from fa/fa rats, with or without 1 nmol/l ulin. (e) Basal or ulin (1 nmol/l) effets on GLUT4 transloation in musle ells from fa/fa rats transfeted with nnosα DNA () or an empty vetor (ontrol). (f) Western blot analysis of GLUT4 expression after transfetion with nnosα DNA () or an empty vetor (ontrol). p <.5; p <.1; p <.1

7 Diabetologia (214) 57: stimulated (1 nmol/l) onditions, respetively (Fig. 6d). Despite the use of α tead of μ in our overexpression experiments, we propose that restoration of expression was able to improve ulin-stimulated gluose utilisation in ulin-resistant skeletal musle. Improvement of GLUT4 transloation by restoration We first found no differenes in GLUT4 expression in musle from fa/fa and fa/+ rats (data not shown). Then, to address the mehanisms by whih restoration improves gluose uptake in musle ells from fa/fa rats, we measured GLUT4 transloation and found a 96% inrease in GLUT4 transport (p <.5, n =3) when was overexpressed in the presene of ulin, but not in basal onditions (Fig. 6e). Interestingly, 1 nmol/l ulin, whih was unable to enhane gluose uptake (see Fig. 4b), stimulated GLUT4 transport by 38% (p <.5, n =3). NO has also been shown to modulate GLUT4 expression in L6 myotubes [28], and we found a 38% inrease in GLUT4 protein level in musle ells from fa/fa rats overexpressing (p <.5) (Fig. 6f). Involvement of a ternary omplex between, Hsp7 and CHIP in proteolyti degradation is known to interat with two haperones, Hsp9 and Hsp7, whih oppositely affet stability: Hsp9 partiipates in inhibition and Hsp7 in stimulation of ubiquitination [29]. Proteasomal degradation of requires ubiquitination by the haperone-dependent E3 ubiquitin ligase, CHIP [23]. Hsp7 and CHIP were found to be expressed in skeletal musle. No differene in Hsp7 expression ould be observed in musles from fa/+ and fa/fa rats (Fig. 7a); in ontrast, CHIP expression was higher in fa/fa vs fa/+ rats (p <.1) (Fig. 7b). Using immunopreipitation experiments, we found an Hsp7 CHIP ternary omplex in skeletal musle. Most importantly, greater amounts of (p <.5) and Hsp7 (p <.5) ould be deteted when CHIP was immunopreipitated from fa/fa rat musles (Fig. 7). Hene, we onlude that proteasomal degradation in the ulin-resistant skeletal musle involves both Hsp7 and the ubiquitin ligase CHIP. Disussion The μ isoform plays a key role in the regulation of ontratile fore and gluose utilisation in skeletal musle [6, 7]. Early studies suggested the presene of defets in the signalling pathway in ulin-resistant musle. More preisely, skeletal musles from Zuker fa/fa rats have been reported to display dereased NOS atalyti ativity [11]. a b 64 kda 36 kda protein level Total lysate Lean fa/ Hsp7 protein level CHIP protein level Obese fa/fa IP CHIP Hsp7 protein level CHIP Hsp7 CHIP Impaired basal and ulin-stimulated musle NOS ativity has also been desribed in ulin-resistant musle from individuals with type 2 diabetes [3]. These observations prompted us to study the mehanisms responsible for an defet in ulin-resistant musle, using the Zuker fa/fa rat as a model. The dereased expression we observed in Zuker fa/fa rats partly aounts for the impaired atalyti ativity. Adereasekeletalmusleexpressionoursina broad panel of gluose homeostasis disorders; it has been found in ZDF as well as in Goto-Kakizaki diabeti rats [31], and in prediabeti and type 2 diabeti individuals [32, 33]. Hsp7 Fig. 7 Evidene for a ternary omplex between, Hsp7 and CHIP in gastronemius musle from Zuker fa/fa vs fa/+ rats. Expression of Hsp7 (a) and CHIP (b) by western blot in musle extrats from fa/+ and fa/fa rats, and relative quantifiation of the band intensities. () Co-immunopreipitation of CHIP with Hsp7 and in musles from fa/+ and fa/fa rats. CHIP was immunopreipitated (IP) and the presene of Hsp7 and was revealed by western blot. Eah western blot is representative of three independent experiments. p <.5; p <.1

8 184 Diabetologia (214) 57: Conerning enos, this isoform has been shown to be expressed in endothelial ells and some skeletal musle fibres [3]. Even though the endothelium-dependent vasodilator response is impaired in ulin-resistant musle [34], we found it unhanged in our study in Zuker fa/fa rat musle, despite previous onfliting data [35, 36]. Proteolyti degradation of NOS an be onsidered as a regulatory mehanism aimed at ontrolling NOS protein quality. In ulin-resistant skeletal musle, we found that was atively degraded by the proteasome [37], as demonstrated by the presene of high amounts of ubiquitinated and by the effet of the inhibitor MG 132, whih was able to restore expression and atalyti ativity to a normal level. We also deteted lower amounts of SDS-resistant dimers in fa/fa vs fa/+ skeletal musle (data not shown), whih might favour inreased suseptibility to proteasomal degradation; indeed, dimerisation is known to protet from proteolysis [38]. The speifi signals targeting to the proteasome have not been learly identified. A reent study reported that Lys739 in the almodulin-binding domain is a site for ubiquitination of in vitro and partiipates in the CHIP-dependent degradation of in HEK293 ells [39]. Based on the UbPred predition [25], we also identified Lys131 as a potential ubiquitination site, in aordane with our observation that the entire undergoes more degradation than splie variants that lak the PDZ domain (Fig. 3). Using a ell-permeant peptide ounterating Lys131 ubiquitination, we partially restored expression, and thereby onfirmed the involvement of the N-terminal part of the enzyme in its proteasomal degradation. Nonetheless, other ubiquitination sites that are present in all variants (e.g. Lys739 [39]) might also partiipate in degradation of the enzyme, but with less important turnover. Prote assigned to degradation are labelled with a multiubiquitin hain and then targeted to the 26S proteasome. Among the ubiquitination enzymes, CHIP is a U-boxontaining E3 ubiquitin ligase that binds to the haperones Hsp7 and Hsp9 [4]. It has been demonstrated that CHIP ubiquitinates and that the quantity of ubiquitin onjugates is greatly enhaned upon the interation of CHIP with -bound Hsp7 [23]. In ontrast, binding of Hsp9 to has been reported to enhane the enzyme satalyti ativity [41], together with almodulin-dependent inhibition of its ubiquitination [29]. We showed that CHIP and Hsp7 are both present in skeletal musle. Hsp7 expression was unhanged and CHIP expression was inreased in fa/fa rats, a finding that ould relate to the enhaned proteolysis and musle wasting observed in ulin-resistant skeletal musle [42]. Indeed, inreased CHIP-dependent proteasomal ativity ourred in fa/fa musle, as pharmaologial blokade with MG 132 and, more interestingly, with Methylene Blue highly improved gluose uptake in ulin-resistant musle but not in ulin-sensitive tissue. Furthermore, our immunopreipitation experiments onfirmed the presene of a ternary omplex between, Hsp7 and CHIP in skeletal musle. Most importantly, the greater assoiation between the three partners in ulin-resistant musle is likely to aount for the inreased proteolyti degradation of. In Zuker rats, we onfirmed that is strongly assoiated with the sarolemma of musle fibres, as already desribed by others [4, 43]. We also deteted a more puntuated staining in the saroplasm, probably orresponding to loalised in intraellular organelles suh as the Golgi omplex [16]. An important observation is the strong derease in sarolemmal loalisation of in skeletal musle from Zuker fa/fa rats. Suh a hange has also been shown to our for in musle fibres from patients with Duhenne musular dystrophy [44] and in the Goto- Kakizaki rat model of type 2 diabetes [31]. From a pathophysiologial point of view, alterations in the signalling pathway resulting from misloalisation might be involved in musle fatigue and atrophy intervening in ulin-resistant skeletal musle [45, 46]. Conerning the expression of the protein inhibitor of, PIN, originally loned in rat brain [18], and identified in ventilatory musles [43] and panreati beta ells [47], we found levels of PIN to be almost unhanged in ulin-resistant musles. In addition, PIN sarolemmal loalisation dereased in line with that of, pointing to a minor role of PIN in alterations. Of major interest is the observation that restoration of expression by bloking degradation or by overexpression was able to improve ulin-stimulated gluose uptake in primary musle ells from fa/fa rats. We are aware of the potential limitations assoiated with the use of α tead of μ in these overexpression experiments, despite the fat that both isoforms share the same ubiquitination sites and display idential atalyti ativity [4]. Very interestingly, fa/fa musle ells retained their sensitivity to NO, as shown by the stimulating effet of the NO donor, SNP, independently of the presene of ulin. Suh a positive effet of NO has also been found in ulin-resistant musle ells derived from individuals with type 2 diabetes [21]. In addition, our observations, as those of several other groups, support the view that NO ontrols gluose transport via ulin-independent signalling pathways [48, 49], inluding ativation of GMP-dependent protein kinase [5, 51] and AMP-ativated protein kinase-α1 [48, 52]. Conerning the possible mehanisms involved, we found that restoration of endogenous in ulin-resistant musle ells greatly improved transloation of GLUT4 at the plasma membrane in the presene of ulin, but not in basal onditions. This implies that normal ativity/expression ould also improve the intri ativity of GLUT4, as previously suggested [53], despite no onsensus view in the literature. In addition, restoration inreased GLUT4 expression,

9 Diabetologia (214) 57: as previously desribed in L6 myoytes [28], whih may partiipate at the greater level of the transporter observed at the plasma membrane. We also found that ulin moderately stimulated GLUT4 transloation in musles ells from fa/fa rats, without any improvement in gluose uptake. Suh a finding suggests that ulin resistane in fa/fa rats ould, at least in part, also result from a defet in GLUT4 ativation. In onlusion, ulin-resistant skeletal musle from Zuker fa/fa rats displayed dereased protein expression and misloalisation, whih ould both aount for the impaired atalyti ativity. The loss of was aused by inreased degradation of the enzyme by the ubiquitin proteasome pathway, mediated by a diret interation with Hsp7 and CHIP. Finally, restoration in ulin-resistant musles should be onsidered as a potential new approah to improve musle gluose homeostasis. Aknowledgements We thank M. 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